Novel nucleic acids and polypeptides

ABSTRACT

The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.

1. TECHNICAL FIELD

The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.

2. BACKGROUND

Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.

Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION

The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.

The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.

The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases. The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. These nucleic acid sequences are designated as SEQ ID NO: 1-172, or 345-516. The polypeptides sequences are designated SEQ ID NO: 173-344, or 517-688. The nucleic acids and polypeptides are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.

The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-172, or 345-516 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-172, or 345-516. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-172, or 345-516 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.

The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-172, or 345-516. The sequence information can be a segment of any one of SEQ ID NO: 1-172, or 345-516 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-172, or 345-516.

A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect fill-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors. Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.

In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-172, or 345-516 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art. In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-172, or 345-516 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-172, or 345-516 ; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-172, or 345-516; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-172, or 345-516. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-172, or 345-516; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in SEQ ID NO: 173-344, or 517-688; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-172, or 345-516; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%,75%, 80%,85%, 90%,95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.

The invention also provides compositions comprising a polypeptide of the invention. Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

The invention also provides host cells transformed or transfected with a polynucleotide of the invention.

The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such process is a mature form. of the protein.

Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.

Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.

The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions. The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.

The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.

The invention also provides methods for the identification of compounds that modulate. (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound the binds to a polypeptide of the invention is identified.

The methods of the invention also provide methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.

The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in Table 2); for which they have a signature region (as set forth in Table 3); or for which they have homology to a gene family (as set forth in Table 4). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.

4. DETAILED DESCRIPTION OF THE INVENTION

4.1 Definitions

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide. According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule. Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.

The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules. The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.

The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells. The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes. The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells. PGCs are the source from which GSCs and ES cells are derived. The PGCs, the GSCs and the ES cells are capable of self-renewal. Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.

The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.

As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.

The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil). Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of MRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NOs:1-20.

Probes may, for example, be used to determine whether specific MRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.

The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-172, or 345-516. The sequence information can be a segment of any one of SEQ ID NO: 1-172, or 345-516 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-172, or 345-516. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 4²⁰ possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segments can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.

Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷4²⁵) times the increased probability for mismatch at each nucleotide position (3×25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.

The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.

The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences. For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence. While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.

The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.

The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules. A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 500 amino acids, more preferably less than 200 amino acids more preferably less than 150 amino acids and most preferably less than 100 amino acids. Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.

The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

The term “translated protein coding portion” means a sequence which encodes for the fill length protein which may include any leader sequence or any processing sequence.

The term “mature protein coding. sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence. The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence. The peptide may have been produced by processing in the cell which removes any leader/signal sequence The mature protein portion may or may not include the initial methionine residue. The methionine residue may be removed from the protein during processing in the cell. The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.

The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.

The term “variant” (or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.

Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. “Insertions” or “deletions” are preferably in the range of about I to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.

The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.

The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems. “Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into MRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional, unit into chromosomal DNA or carry the recombinant transcriptional unit extra chromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers. Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eulcaryotic.

The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum. “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2): 134-143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol. 16:27-55)

Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.

The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×X SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.). Other exemplary hybridization conditions are described herein in the examples.

In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity. Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.

The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.

The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration. The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed. The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.

As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.

Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.

4.2 Nucleic Acids of the Invention

Nucleotide sequences of the invention are set forth in the Sequence Listing.

The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-172, or 345-516; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 173-344, or 517-688; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 173-344, or 517-688. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-172, or 345-516; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing as SEQ ID NO: 173-344, or 517-688; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 173-344, or 517-688. Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof, domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.

The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.

The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. Further 5′ and 3′ sequence can be obtained using methods known in the art. For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-172, or 345-516 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-172, or 345-516 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO: 1-172, or 345-516 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.

The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.

The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.

Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-172, or 345-516, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.

The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof Allelic and species variations can be routinely determined by comparing the sequence provided SEQ ID NO: 1-172, or 345-516, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-172, or 345-516 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.

The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-172, or 345-516, can be obtained by searching a database using an algorithm or a program. Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.

Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.

The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.

The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.

In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelnan et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.

A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.

Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.

The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.

In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-172, or 345-516, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.

A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.

The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-172, or 345-516or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-172, or 345-516 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 44854490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudoinonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., U.S.A.). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Polynucleotides of the invention can also be used to induce immune responses. For example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.

4.3 Antisense Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-172, or 345-516, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 173-344, or 517-688 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-172, or 345-516 are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-172, or 345-516), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).

4.4 Ribozymes and Pna Moieties

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-172, or 345-516). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SECX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, SECX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g, Bartel et al., (1993) Science 261:1411-1418.

Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y Acad Sci. 660:27-36; and Maher(1992) Bioassays 14: 807-15.

In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.

PNAs of the invention can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).

In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/098 10) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.

4.5 Hosts

The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences. See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, New York (1989), the disclosure of which is hereby incorporated by reference.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluytveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCTIUS92/09627 (WO93/09222) by Selden et al.; and International Application No. PCTIUS90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.6 Polypeptides of the Invention

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 173-344, or 517-688 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-172, or 345-516 or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-172, or 345-516 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 173-344, or 517-688 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 173-344, or 517-688 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 173-344, or 517-688.

Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.

The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins. The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences. The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form. Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.

Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.

A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown. For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.

In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology. Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.

The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g:, ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 173-344, or 517-688.

The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.

The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example. modifications, in the peptide or DNA sequence, can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acid(s) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.

Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the present invention.

The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i. e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearltm or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.”

The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability. Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.

4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity

Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp.3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1), pp. 320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).

4.7 Chimeric and Fusion Proteins

The invention also provides chimeric or fusion proteins. As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide. Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention. In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention. In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be fused to the N-terminus or C-terminus.

For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein. In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.

In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo. The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.

A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.

4.8 Gene Therapy

Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol.392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.

Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.

The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.

Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thyrnidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.9 Transgenic Animals

In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotidcs of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide. Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.

In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No 5,489,743 and PCT Publication No. WO94/28 122, incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

4.10 Uses and Biological Activity

The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment. Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.

The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.

4.10.1 Research Uses and Utilities

The polynucleotides provided by the present invention can be used by the research community for various purposes The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

4.10.2 Nutritional Uses

Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

4.10.3 Cytokine and Cell Proliferation/Differentiation Activity

A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAIG, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following:

Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lyrnphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Talcai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-y, Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

4.10.4 Stem Cell Growth Factor Activity

A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells. Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors. The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.

It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Fit-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).

Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells. Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium. Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat. No. 5,690,926).

Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential MRNA to create eDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.

Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions. For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders. The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue. In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.

Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types. A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive. For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.

In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).

4.10.5 Hematopoiesis Regulating Activity

A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

Therapeutic compositions of the invention can be used in the following:

Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

4.10.6 Tissue Growth Activity

A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.

A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells. Treatment of osteoporosis, osteoarhritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention.

Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation. Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.

Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate. A polypeptide of the present invention may also exhibit angiogenic activity.

A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

Therapeutic compositions of the invention can be used in the following:

Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).

Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dernatol 71:382-84 (1978).

4.10.7 Immune Stimulating or Suppressing Activity

A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or energy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant Moreover, a lack of costimulation may also be sufficient to energize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/1pr/1pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Up regulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Up regulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β₂ microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zarnai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Anticaet al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

4.10.8 Activin/Inhibin Activity

A polypeptide of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.

The activity of a polypeptide of the invention may, among other means, be measured by the following methods.

Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.-USA 83:3091-3095, 1986.

4.10.9 Chemotactic/Chemokinetic Activity

A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

Therapeutic compositions of the invention can be used in the following:

Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al-Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.

4.10.10 Hemostatic and Thrombolytic Activity

A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).

Therapeutic compositions of the invention can be used in the following:

Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467474, 1988.

4.10.11 Cancer Diagnosis and Therapy

Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.

Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness. Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.

Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer. Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.

The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16 -213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexarnethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g. exposure to carcinogens) known in the art that predispose an individual to developing cancers.

Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.

In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 2 1), tumor systems in nude mice as described in Giovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.

4.10.12 Receptor/Ligand Activity

A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions. A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

The activity of a polypeptide of the invention may, among other means, be measured by the following methods:

Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s). Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.

Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands. The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, calorimetric molecules or a toxin molecules by conventional methods. (“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14 . Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited, to ricin.

4.10.13 Drug Screening

This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.

Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.

Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.

The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof For a review, see Science 282:63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Domer et al., BioorgMed Chem, 4(5):709-15 (1996) (alkylated dipeptides).

Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.

4.10.14 Assay for Receptor Activity

The invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor. The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention. There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention. Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) are then compared. Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.

The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined. For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell. The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e. phosphorylation. Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.

4.10.15 Anti-Inflammatory Activity

Compositions of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material. Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.

4.10.16 Leukemias

Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

4.10.17 Nervous System Disorders

Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:

(i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;

(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;

(iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;

(iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;

(v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;

(vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

(vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and

(viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:

(i) increased survival time of neurons in culture;

(ii) increased sprouting of neurons in culture or in vivo;

(iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or

(iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:1742); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

4.10.18 Other Activities

A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

4.10.19 Identification of Polymorphisms

The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately. For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.

Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed. Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms. The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention. In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.

Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.

4.10.20 Arthritis and Inflammation

The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PBS only.

The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis. of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.

4.11 Therapeutic Methods

The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but are not limited to, those exemplified herein.

4.11.1 EXAMPLE

One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention. While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus. The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.0 μg/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.

4.12 Pharmaceutical Formulations and Routes of Administration

A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.

The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects. Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site). Techniques for formulation arid administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

4.12.1 Routes Of Administration

Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.

4.12.2 Compositions/Formulations

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifing, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.

When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuff or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employee Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylarnine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patients age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

4.12.3 Effective Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

4.12.4 Packaging

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

4.13 Antibodies

Also included in the invention are antibodies to proteins, or fragments of proteins of the invention. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (1g) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_(ab), F_(ab)′ and F_((ab)2) fragments, and an F_(ab) expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG₁, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as inmmunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 173, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Some of these antibodies are discussed below.

4.13.1 Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

4.13.2 Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

4.133 Humanized Antibodies

The antibodies directed against the protein antigens of the invention can further comprise 4 humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

4.13.4 Human Antibodies

Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp 77-96).

In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al,(Nature Biotechnology, 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intem. Rev. Immunol. 13 65-93 (1995)).

Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete filly human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

4.13.5 F_(ab) Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_(ab) fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F_((ab)2) fragment produced by pepsin digestion of an antibody molecule; (ii) an F_(ab) fragment generated by reducing the disulfide bridges of an F_((ab)2) fragment; (iii) an F_(ab) fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F_(v) fragments.

4.13.6 Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Mistein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

4.13.7 Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

4.13.8 Effector Function Engineering

It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

4.13.9 Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succininidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diusocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.

4.14 Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tapc; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention. As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

By providing any of the nucleotide sequences SEQ ID NO: 1-172, or 345-516 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-172, or 345-516 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.

As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

4.15 Triple Helix Formation

In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an MRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

4.16 Diagnostic Assays and Kits

The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.

In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

4.17 Medical Imaging

The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.

4.18 Screening Assays

Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-172, or 345-516, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:

(a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and

(b) determining whether the agent binds to said protein or said nucleic acid.

In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.

Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.

Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the MRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.

4.19 Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-172, or 345-516. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-172, or 345-516 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in U.S. Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both. The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of MRNA probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as 17 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.

4.20 Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers. Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol. 28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlenet al., 1987; Morrissey& Collins, (1989) Mol. Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad. Sci. USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used. Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLinkNH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling. CovaLink. Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussenet al., (1991) Anal. Biochem. 198(1) 13842).

The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11 (8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.

More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm₇), is then added to a final concentration of 10 mM 1-MeIm₇. A ss DNA solution is then dispensed into CovaLinkNH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm₇, is made fresh and 25 ul added per well. The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.

An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73, incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal. Biochem. 169(1) 104-8; all references being specifically incorporated herein.

To link an oligonucleotide to a nylon support, as described by VanNess et al. (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.

One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994)PNAS USA 91(11) 5022-6, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.

4.21 Preparation of Nucleic Acid Fragments

The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or CDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.

The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6, incorporated herein by reference). In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al. (1992) Nucleic Acids Res. 20(14)3753-62. These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.

The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small moleculepUC19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI* * digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI* * restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.

As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization. This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.

4.22 Preparation of DNA Arrays

Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm², depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane. subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm² and there may be a 1 mm space between subarrays.

Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.

The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.

All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.

5. EXAMPLES 5.1 Example 1

Novel Nucleic Acid Sequences Obtained from Various Libraries

A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts. Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers)to obtain signature sequences. The clones were clustered into groups of similar or identical sequences. Representative clones were selected for sequencing.

In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences. In some cases RACE (Random Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.

5.2 Example 2

Assemblage of Novel Nucleic Acids

The contigs or nucleic acids of the present invention, designated as SEQ ID NO: 345-516 were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 114, gb pri 114, and UniGene version 101 ) that belong to this assemblage The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.

Table 6 sets forth the novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 517-688) of the present invention, and their corresponding nucleotide locations to each of SEQ ID NO: 517-688. Table 6 also indicates the method by which the polypeptide was predicted. Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fasta.bioch.viriniaedu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R. Pearson, Methods in Enzymology, 1 83:63-98 (1990), herein incorporated by reference). Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol. Biol., 268:78-94 (1997), incorporated herein by reference). Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.

5.3 Example 3

Novel Nucleic Acids

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), full-length gene cDNA sequences and their corresponding protein sequences were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genebank. Other computer programs which may have been used in the editing process were phredPhrap and Consed ((University of Washington) and ed-ready, ed-ext and gc-zip2 (Hyseq, Inc.)). The full-length nucleotide sequences are shown in the Sequence Listing as SEQ ID NO: 1-23. The amino acids are SEQ ID NO: 173-195.

Table 1 shows the various tissue sources of SEQ ID NO: 1-23.

The nearest neighbor results for SEQ ID NO: 1-23 were obtained by a BLASTP (version 2.0al 19MP-WashU) search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-23 from Genpept. The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologs with identifiable functions for SEQ ID NO: 1-23 are shown in Table 2 below.

Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp. Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.

Using the pFam software program (Sonnhanuner et al., Nucleic Acids Res., Vol. 26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4 shows the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.

The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine from using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifing prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites” Protein Engineering, Vol. 10, no. 1, pp. 1-6 (1997), incorporated herein by reference. A maximum S score and a mcan S score, as described in the Nielson et as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.

5.4 Example 4

Novel Nucleic Acids

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full length gene cDNA sequence and its corresponding protein sequence were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genbank (i.e. dbEST version 1 17, gb pri 117, UniGene version 117, Genpept release 117). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS: 24-137. The corresponding amino acids are SEQ ID NO: 196-309.

Table 1 shows the various tissue sources of SEQ ID NO: 24-137.

The nearest neighbor results for SEQ ID NO: 24-137 were obtained by a BLASTP version 2.0al 19MP-WashU search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the closest homologue for SEQ ID NO: 24-137 from Genpept. The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologs with identifiable functions for SEQ ID NO: 24-137 are shown in Table 2 below.

Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp. Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.

Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4 shows the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.

The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine from using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites” Protein Engineering, Vol. 10, no. 1, pp. 1-6 (1997), incorporated herein by reference. A maximum S score and a mean S score, as described in the Nielson et as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.

5.5 Example 5)

Novel Nucleic Acids

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length gene cDNA sequence and its corresponding protein sequence were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genbank (i.e., dbEST version 118, gb pri 118, UniGene version 118, Genpept release 118). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS: 138-172. The corresponding amino acid sequences are SEQ ID NO: 310-344.

Table 1 shows the various tissue sources of SEQ ID NO: 138-172.

The homology results for SEQ ID NO: 138-172 were obtained by a BLASTP version 2.0al 19MP-WashU search against Genpept release 121 and Geneseq release 200101 (Derwent), using BLAST algorithm. The nearest neighbor result showed the homologs for SEQ ID NO: 138-172 from Genpept. The translated amino acid sequences for which the nucleic acid sequence encodes are shown in the Sequence Listing. The homologues with identifiable functions for SEQ ID NO: 138-172 are shown in Table 2 below.

Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp. Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.

Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4 shows the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.

The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine from using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites” Protein Engineering, Vol. 10, no. 1, pp. 1-6 (1997), incorporated herein by reference. A maximum S score and a mean S score, as described in the Nielson et as reference, was obtained for the polypeptide sequences. Table 7 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.

Table 5 is a correlation table of all of the sequences and SEQ ID Nos. TABLE 1 Tissue Origin RNA Source Library Name SEQ ID NOS: fibroblast Strategene LFB001 37 62-63 69 87-89 108 124 126-127 144-145 166 168 NULL NULL CGSd001 25 54 74 NULL NULL CGSd004 26 80 NULL NULL CGSd005 11 91 166 NULL NULL CGSd006 11 25-26 34 39 60 139 143 160 164 166 NULL NULL CGSd009 13 45 NULL NULL CGd007 34 NULL NULL CGd008 34 39 76 NULL NULL CTL016 170 NULL NULL CTL021 57 76 78 NULL NULL PCR2V1 1 57 95 NULL NULL PGEMV1 10 26 32 34 42 44 52 57 62 70 78 95 113-114 122-124 144-145 150 NULL NULL SUP002 32 35 57 62 78 149 153-154 168 NULL NULL TEST 52 adipocytes Stratagene ADP001 6 8 38 91 95 97 104 108 110 144-145 153-154 167 adrenal Clontech ADR002 6 9 14 16 22 27 38-39 41 45 52 54 gland 62-63 74 79 81 90 107 111 123 126 128 136 166 168 adult BioChain ABR012 38 126 brain adult BioChain ABR013 35 53 brain adult Clontech ABR001 15 17 22 35 72 84 126 brain adult Clontech ABR006 11 22-23 31-32 39 43 52 54-55 60 brain 81 84 102 110 139 149 168 adult Clontech ABR008 1-4 7-12 18 22-24 27-28 31-33 35 brain 40 44-45 50 52-53 55 57-60 62 72 74 81 84 87-89 93-96 98-99 102 104 107-108 110-111 113-114 120-123 130-131 136 142-145 147-148 150 157 160 164 167-168 171-172 adult Clontech ABR011 23 38 brain adult GIBCO AB3001 6 17 19 22 38 52 54-55 62 74 86 brain 104 122 127-128 131 164 adult GIBCO ABD003 6 10 18 22-23 32 35 38 40 52-53 brain 55 58 62 71-72 74 79-81 94-95 99 105 108 111 113-114 122 126 128 130-131 139 142 160-161 167-169 adult Invitrogen ABR014 57 62 76 78 96 120-121 161 brain adult Invitrogen ABR015 6 37 76 91 99 brain adult Invitrogen ABR016 57 63 brain adult Invitrogen ABT004 10 23 32 40 43 55 58 60 74 84 99 brain 112 120-121 148 167-168 adult GIBCO AHR001 2 6 14 18-19 22 30 38-39 45 51-54 heart 57 60 62-63 66-67 74 76 79 81 87-91 93-95 98 104 108 110 113-114 122-124 126-128 135-136 140-141 144-145 150 153-154 160 163 168-169 171-172 adult GIBCO AKD001 2 6-10 14 17-19 22 25 28-29 33-35 kidney 38-39 42-45 52 54-55 58 60-63 67 72 74 79-80 86-89 91-96 99-101 104-105 107-108 111-114 116 122-124 126-128 130-132 136 138-139 141 144-145 147 155 160 167-168 172 adult Invitrogen AKT002 1-2 6 20 34 38 41 43 52 62 76 81 kidney 87-90 94-95 105 138 152-154 160 adult Clontech ALV003 38 57 76 95 149 liver adult Invitrogen ALV002 9 14 22 26 41-42 45 55 62 65 87-89 liver 97 111 120-121 141 149 160 162 164 168 adult lung GIBCO ALG001 38 41 54-55 57 62 76 79 81 86 91 104 111-112 120-121 123 160 163 adult lung Invitrogen LGT002 1-3 5 8 10 14 17 19 21-23 25 27 29 34 37-38 40-41 44-45 52-54 60-62 68-70 81 84 86-91 93-96 99-101 104 106-108 112-114 122-123 127 131-132 135-136 141 144-145 148 152-157 160-162 165 167-168 171-172 adult Clontech SPLc01 9 16 52 55 62 81 143 150 153-154 spleen adult GIBCO ASP001 1-2 6 10 18 34 38 40 42 51 57 61 spleen 72 74-76 78 80-81 86 90-91 95 104 108 110-111 122-123 126 130-131 143 153-154 168 172 bladder Invitrogen BLD001 1 15 18 34 51 87-89 95 97 132 164 bone Clonetech BMD007 57 76 138 marrow bone Clontech BMD001 1-3 6 8-11 13-14 19 21 31 36 38-41 marrow 50 53-54 57 60-62 69 74 76 78 81 85 90-91 93-96 105 107-109 112-114 122 125-128 139 141 143 148 153-154 159 161-162 166 169-172 bone Clontech BMD004 38 57 76 91 98 marrow bone GF BMD002 9 12 16 19 23 25 27 39 41-44 52-59 marrow 62 73-74 76 78 81 84 91 94-99 109-110 113-114 120-121 125-128 131 135 137 139 141-143 150 153-154 162 168 172 cervix BioChain CVX001 2 6-7 10 17 19-20 23-24 27 34 38 52 54 60 62-63 69 80 87-91 93-96 100-101 108 111 113-114 123 127-128 134 136 141 144-145 152-154 158 162 165 colon Invitrogen CLN001 23-24 55 65 72 95 120-123 142 171 diaphragm BioChain DIA002 62 126 153-154 endothelial Strategene EDT001 1 6-7 10-11 14 16-19 22 24 27 35 cells 37-40 42-43 47 49 52 58 60 62-63 67 69 72 74 81 90-91 93-101 104 107-108 120-124 126-133 135-136 141-142 144-145 151 153-154 156 160 165 167-168 170-172 esophagus BioChain ESO002 23 fetal Clontech FBR001 60 91 brain fetal Clontech FBR004 1 3 11 22 32 74 brain fetal Clontech FBR006 1 9-12 14 16 22 29 32-33 36 40 42 brain 45 57 59-60 72 80-81 84 91 94 97-98 108 111 113-114 119-123 135-137 143-145 147-148 157 160 166 169 171-172 fetal GIBCO HFB001 1-2 6-7 10 13 22-23 27-28 32 37-39 brain 42 44 48 51-52 54 57-58 60 62 69 72 76 78-80 91 93-95 102 108 112-114 120-123 126 128 130-132 135 138-139 144-145 152 155 160-161 164 167 fetal Invitrogen FBT002 1 3 8 22-23 40 42 62 74 78 80 84 brain 91 95 98 100-101 108 122 124 136 142-145 147 160 fetal Invitrogen FHR001 57 160 heart fetal Clontech FKD001 7 38 40 60 62 85 87-89 95 147 kidney fetal Clontech FKD002 42 95 kidney fetal Invitrogen FKD007 57 62 78 91 95 kidney fetal Clontech FLV004 41 44 57 62 69 72 95 111 149 169 liver fetal Invitrogen FLV001 3 18 22 27 34 52 55 57-58 76 78 liver 87-89 91 111 120-121 123 131 136 144-145 149 168 170 fetal Soares FLS001 1-3 5-6 10-11 13-14 16-19 23-24 liver- 26-27 29 31 34-35 38-44 46 50-52 spleen 54 57-65 67-70 72 74 76 78-81 84 86-91 93-98 103-104 106 108 111-112 117 120-124 126-128 130-133 135-136 138 141 143-145 147 149 155-156 160-162 165 167-172 fetal Soares FLS002 3 5-6 8-10 13 16-19 23-24 26-27 liver- 34-35 37 41 44-45 50-51 53-55 57-58 spleen 62-64 68-70 74-75 79 81 84 87-89 91 93-94 96 98-99 103-105 108 111-115 117 119 123-124 126-128 131-132 134-136 143-148 150 153-154 156 161-162 164 168 170-172 fetal Soares FLS003 26 50 53 57 62 76 78 96 149-150 liver- spleen fetal lung Clontech FLG001 94-95 115 131 149 168 fetal lung Invitrogen FLG003 1 24 40 57 62 73 76 78 80-81 95 111 136 153-154 168 fetal Invitrogen FMS001 24 27 30 54-58 90 94 99 119 123 muscle 131 140 142 147 164 168 fetal Invitrogen FMS002 42 122 155 muscle fetal skin Invitrogen FSK001 1 6 18 22-24 27 43 45 52 55 57 60 62 68-69 78-79 81 91 94-95 97 99 108 112-114 120-121 142 147 153-154 161 164-165 167-170 fetal skin Invitrogen FSK002 1 29 40-41 54-55 62 69 94 96 115 153-154 fetal BioChain FSP001 91 95 spleen infant NULL IBM002 3 11 23 32 128 brain infant NULL IBS001 44 59 120-121 170 brain infant Soares IB2002 3 10-13 18-19 22-23 25 27 32 37-40 brain 43 54-55 57 59-63 77-78 84 94 98-99 102 105 108 120-121 123-124 127 130 136-137 143-145 152 155-156 160 164 167-168 170 infant Soares IB2003 1 3 23 27 37 40 51 60 62 72 99 brain 104 108 111 120-123 128 136 139 143-145 167 leukocytes Clontech LUC003 1-2 38 62 76 84 94 97 128 136 161 165 172 leukocytes GIBCO LUC001 2 6-8 11 13-14 16 18-19 22-24 27-28 37-38 44-45 52-53 57-58 60 62 67 69-70 74 76 78-81 84 90-91 93-95 97-101 107-114 122-123 126-128 130-132 135-136 138-139 141 143 147 156-157 161-162 165-167 lung 37 62-63 69 87-89 108 124 126-127 144-145 166 168 lung Strategene LFB001 1-2 6-7 10 14 19 lymph node Clontech ALN001 6 19 34 39 62 80 90 93 95 112-114 127 143 162 lymphocyte ATCC LPC001 37-38 43 51 55 57 62 70 72 76 78-79 90 94-95 97 108 112 119 122 132 143 146 165-166 macrophage Invitrogen HMP001 113-114 mammary Invitrogen MMG001 1-2 8 13-14 18 22-23 27 33 40 43 gland 52 55 58-60 70 74 84 86-90 93 95 97 99-101 104 107-108 111 113-114 120-121 123 131 136 142-145 147 149 153-154 160 165 167-169 172 melanoma Clontech MEL004 2 6 40 42 51 60 62-63 67 74 91 100-101 107 112 136 161 168 170 mix NULL SUP005 26 40 52 57 62 78 95 149 153-154 mix NULL SUP008 1 57 78 84 120-121 mix NULL SUP009 26 40 44 57 76 78 144-145 153-154 mixed EST CGd010 34 mixed NULL CGd012 11 25-26 31 33-34 39 75 147 157 163 mixed NULL CGd013 26 mixed NULL CGd015 25-26 39 44 47 52 148 153-154 160 mixed NULL CGd016 147 neuron Strategene NTD001 6 23 47 49 60 74 81 94-95 113-114 129-130 133 135 138 151 166 neuron Strategene NTR001 6 59 91 95 128 136 148 neuronal Strategene NTU001 14 23 55 59 95 100-101 108 111-112 cells 122 132-133 136 151 168 170 ovary Invitrogen AOV001 1-3 6-9 11 14 16-19 21-23 27-29 37-38 40-45 51-52 54 58-60 62-63 67 72 74-76 79 81 86-91 93-97 100-101 103 105 107-108 111-115 119-124 126-128 130-132 135 138 141 144-145 147 150 152-156 160-161 165 167-169 172 pituitary Clontech PIT004 2 34 57 62 74 78 gland placenta Invitrogen APL001 42 59 95 148 placenta Invitrogen APL002 24 33-34 62 76 87-90 95 99 122 131 136 143 165 167 prostate Clontech PRT001 6 17 37-38 54 62-63 65-66 72 76 87-89 97 105 160 172 rectum Invitrogen REC001 22 24 34 54 95 97 122 168 salivary Clontech SAL001 6 59 87-89 91 107-108 131 138 gland 144-145 150 160 skeletal Clonetech SKMS03 153-154 muscle skeletal Clontech SKM001 1 56-57 81 86 90-91 94 123 153-155 muscle skeletal NULL SKMS04 38 muscle skin ATCC SFB001 72 95 fibroblast skin ATCC SFB003 62 95-96 fibroblast small Clontech SIN001 2 25 33 37-40 54 62 65 70 74 80-81 intestine 84 86 90 136 143 153-155 160 164 168-169 172 spinal Clontech SPC001 3 6 12 37-39 55 57-58 70 74 76 84 cord 91 94-95 98 104 113-114 126 132 138 157 160 168 stomach Clontech STO001 1 25 33 35 39 43 74 95 132 testis GIBCO ATS001 1 14 17-18 38 44 46 60 74 76 81 91 94-95 111 115 126 128 132 139 152-154 156 160 168-169 172 thalamus Clontech THA002 5 18 22 24 41 43 52 65 84 99 104 110 123 144-146 158 thymus Clonetech THM001 6 9 18-19 34 38 41-42 45 52-54 63 69 84 94-96 99 112 127-228 130-131 141 160 170 172 thymus Clontech THMc02 1 3 7 9 16 23 27 35 40 44 51-53 62-63 72 81 84 86-90 94-95 99-101 115 123 128 132 141 143-145 148 167 169-171 thyroid Clontech THR001 1 3 6 16-19 22 29 35 38 42 45 52 gland 54 56 58 60-63 74 76 81 84 87-89 91 93-95 97 99-101 105 107-108 111 113-114 122-123 126-128 131 135-136 141 144-145 147 160-162 164-165 168 172 trachea Clontech TRC001 5 69 76 80 90-91 93 113-114 166 umbilical BioChain FUC001 6-7 10-11 14 22 29 41 43 51 57 62 cord 74 76 78 80-81 91 94 97 105 110 115 124 131-132 141 144-145 150 160 165 168-169 171-172 uterus Clontech UTR001 37 52 67 76 81 95 108 112 118 142 168 young GIBCO ALV001 2 26 29 38 52 54 63 72 74 95-97 liver 99 104 108 111 120-121 123 149 156 164 169 172

TABLE 2 SEQ SMITH- ID ACCESSION WATERMAN NO: NUMBER SPECIES DESCRIPTION SCORE % IDENTITY 1 W80293 Homo sapiens Human translocation 1016 96 associated protein designated Gp25L-H. 2 M74089 Homo sapiens TB1 2226 99 3 D88827 Homo sapiens zinc finger protein FPM315 447 46 4 Z34818 Homo sapiens voltage-dependent L-type 1363 96 Ca channel alpha 1 subunit 5 U60269 Homo sapiens putative envelope protein; 370 98 orf similar to env of Type A and Type B retroviruses and to class II HERVs 6 Z47087 Homo sapiens RNA polymerase II 865 100 elongation factor-like protein 7 AL078461 Homo sapiens dJ901O8.1 (continues in 782 97 dJ1121G12 (AL109965)) 8 X93357 Mus musculus homolog of human SYT 870 53 9 AF227156 Homo sapiens RRN3 1679 98 10 AC004908 Homo sapiens similar to ribosomal protein 377 94 L23a; similar to P29316 (PID: g132848) 11 U10556 Saccharomyces Yhr085 wp 175 31 cerevisiae 12 M29581 Homo sapiens zinc finger protein 8 (ZFP8) 2949 99 13 M64788 Homo sapiens GTPase activating protein 1235 55 14 AK026016 Homo sapiens unnamed protein product 1846 100 15 G00637 Homo sapiens Human secreted protein, 166 64 SEQ ID NO: 4718. 16 AF201941 Homo sapiens DC8 1226 100 17 Z83826 Homo sapiens dJ473B4.1 (novel protein 1083 100 similar to predicted human and worm genes) 18 AB031052 Homo sapiens vaccinia related kinase 3 2521 100 19 AL050091 Homo sapiens hypothetical protein 1011 100 20 AF090931 Homo sapiens PRO0483 137 65 21 U39320 Rattus norvegicus cysteine string protein 747 68 22 Y66718 Homo sapiens Membrane-bound protein 1554 70 PRO1106. 24 AE003612 Drosophila CG12393 gene product 244 36 melanogaster 25 V00662 Homo sapiens ATPase 6 232 100 26 T12920_cd1 Homo sapiens 11-AUG-1994 Human 731 100 serum albumin gene with restriction site between domains 2 and 3. 27 AL109804 Homo sapiens dJ1009E24.3 (A novel 920 100 protein) 28 AL021918 Homo sapiens b34I8.1 (Kruppel related 649 54 Zinc Finger protein 184) 29 G01131 Homo sapiens Human secreted protein, 666 99 SEQ ID NO: 5212. 30 Y73373 Homo sapiens HTRM clone 921803 1225 100 protein sequence. 31 AJ293573 Homo sapiens zinc finger protein Cezanne 2243 100 32 AF157634 Homo sapiens collapsin response mediator 2943 99 protein-5 33 J00287 Homo sapiens pepsinogen 2032 100 34 AF264014 Homo sapiens scavenger receptor cysteine- 4523 99 rich type 1 protein M160 precursor 35 AK024433 Homo sapiens FLJ00023 protein 918 100 36 AF119855 Homo sapiens PRO1847 253 71 37 AF176667 Xenopus laevis F-box protein 28 426 80 38 M85148 Macaca mulatta cytochrome oxidase subunit 178 68 III 39 R83119 Homo sapiens Human cisplatin resistance 396 98 protein. 40 X58521 Homo sapiens nucleoporin p62 2068 99 41 Y86211 Homo sapiens Nuclear transport protein 1432 87 clone hfb066 protein sequence. 42 AL035587 Homo sapiens dJ475N16.3 (novel protein 1243 99 similar to RPL7A (60S ribosomal protein L7A)) 43 AF233522 Homo sapiens gamma-adaptin related 3196 100 protein, GGA2 44 AF151066 Homo sapiens HSPC232 1513 93 45 AF113539 Homo sapiens hypothalamus protein 1647 99 HT001 46 AF090901 Homo sapiens PRO0195 120 60 47 J02459 bacteriophage D (head-DNA 561 99 lambda stabilization; 110) 48 AF068294 Homo sapiens HDCMB45P 234 72 49 J02459 bacteriophage R (cell lysis; 158) 834 100 lambda 50 U71363 Homo sapiens zinc finger protein zfp6 396 47 51 AL022393 Homo sapiens p373c6.1 2751 99 52 AF257330 Homo sapiens COBW-like protein 1188 99 53 Y07010 Homo sapiens Breast cancer associated 767 98 antigen precursor sequence. 54 AL158056 Schizosaccharomyces similar to yeast Ecm29 cell 813 25 pombe wall stucture/biosynthesis protein 55 Y44559 Homo sapiens Human Rhotekin protein. 2721 100 56 D86639 Mus musculus stac 360 38 57 V00488 Homo sapiens alpha globin 382 98 58 AL033378 Homo sapiens dJ323M4.1 (KIAA0790 6030 100 protein) 59 AB026111 Homo sapiens XAB2 4470 99 60 AB043550 Mus musculus neural activity-related ring 3852 99 finger protein 61 D38112 Homo sapiens ATPase subunit 6 260 98 62 X15187 Homo sapiens precursor polypeptide (AA - 4112 100 21 to 782) 63 D50369 Homo sapiens low molecular mass 383 98 ubiquinone-binding protein 64 AF161356 Homo sapiens HSPC093 126 58 65 Z11502 Homo sapiens intestine-specific annexin 1426 99 66 J03941 Mus musculus ferritin heavy chain 962 100 67 AB034206 Homo sapiens MCT-1 962 100 68 AF118086 Homo sapiens PRO1992 107 58 69 AF182416 Homo sapiens MDS015 1739 97 70 G03240 Homo sapiens Human secreted protein, 108 56 SEQ ID NO: 7321. 71 G00397 Homo sapiens Human secreted protein, 161 62 SEQ ID NO: 4478. 72 X76538 Homo sapiens hMpv17 569 100 73 AC005626 Homo sapiens R29124_1 1199 97 74 G01332 Homo sapiens Human secreted protein, 407 87 SEQ ID NO: 5413. 75 Y53883 Homo sapiens A suppressor of cytokine 1250 100 signalling protein designated HSCOP-3. 76 M34059 Homo sapiens beta-globin 962 100 78 V00488 Homo sapiens alpha globin 733 100 79 Z11907_cd1 Homo sapiens 19-JAN-1999 Human 1282 100 potassium channel K + Hnov28 cDNA (5′ splice variant 1). 80 U47924 Homo sapiens C10 627 100 81 AF134894 Homo sapiens SWAP-70 homolog 3019 100 82 AB042027 Mus musculus Golgi-associated band 4.1- 4806 93 like protein 83 G03793 Homo sapiens Human secreted protein, 209 71 SEQ ID NO: 7874. 84 X83301 Homo sapiens SMA5 754 97 85 V00662 Homo sapiens URF 2 (NADH 211 91 dehydrogenase subunit) 86 W88627 Homo sapiens Secreted protein encoded by 230 69 gene 94 clone HPMBQ32. 87 Y27607 Homo sapiens Human secreted protein 1461 100 encoded by gene No. 41. 88 Y27607 Homo sapiens Human secreted protein 1340 94 encoded by gene No. 41. 89 Y94295 Homo sapiens Human coenzyme A- 1068 100 utilising enzyme CoAEN-3. 90 AL080169 Homo sapiens hypothetical protein 973 98 91 D14531 Homo sapiens ‘human homologue of rat 974 100 ribosomal protein L9’ 93 AC006465 Homo sapiens supported by mouse EST 437 100 AA277724 (NID: g1917632) and Genscan 94 AL137798 Homo sapiens dJ1182A14.5.1 (novel gene 2075 94 (isoform 1)) 95 M77232 Homo sapiens ribosomal protein S6 706 97 96 AF261717 Homo sapiens SAR1 1032 100 97 X06256 Homo sapiens integrin alpha 5 subunit 5541 100 precursor 98 AC004472 Homo sapiens P1.11659_5 542 98 99 W85459 Homo sapiens Secreted protein encoded by 1650 98 clone dh1135_9. 100 AF201333 Homo sapiens delta-tubulin 2380 100 101 AF201333 Homo sapiens delta-tubulin 1345 100 102 AB036836 Homo sapiens Carbonic anhydrase-related 1740 100 protein 10 104 W80748 Homo sapiens Human mitochondrial 1102 100 chaperone protein (Hmt- GrpE). 105 G00376 Homo sapiens Human secreted protein, 130 73 SEQ ID NO: 4457. 106 Y99444 Homo sapiens Human PRO1575 1241 94 (UNQ781) amino acid sequence SEQ ID NO: 358. 107 G03787 Homo sapiens Human secreted protein, 232 74 SEQ ID NO: 7868. 108 AJ237734 Homo sapiens ribophorin II 2619 100 110 Y99349 Homo sapiens Human PRO1110 1683 100 (UNQ553) amino acid sequence SEQ ID NO: 31. 111 AF258341 Homo sapiens NADPH-cytochrome P450 3540 99 reductase 112 AB044547 Homo sapiens heterogeneous nuclear 3178 99 ribonucleoprotein L 113 AF067804 Homo sapiens HDCMC04P 2124 94 114 AF067804 Homo sapiens HDCMC04P 2219 97 115 U73778 Homo sapiens collagen type XII alpha-1 16043 99 116 G02922 Homo sapiens Human secreted protein, 120 69 SEQ ID NO: 7003. 118 G03133 Homo sapiens Human secreted protein, 175 59 SEQ ID NO: 7214. 119 AF115345 Homo sapiens calcium-regulated heat 282 100 stable protein CRHSP-24 120 AF265228 Homo sapiens DNMT1 associated protein-1 2422 100 121 AF265228 Homo sapiens DNMT1 associated protein-1 1320 85 122 AB018189 Xenopus laevis NO27 907 76 123 AB033603 Homo sapiens cop9 complex subunit 7a 1364 100 124 AL121805 Drosophila BACN4L24.f 200 32 melanogaster 125 Y03000 Homo sapiens Fragment of human secreted 153 72 protein encoded by gene 121. 126 D38112 Homo sapiens NADH dehydrogenase 448 90 subunit 4L 127 AL050091 Homo sapiens hypothetical protein 1011 100 128 AK023702 Homo sapiens unnamed protein product 2774 100 129 J02459 bacteriophage M (tail component; 109) 578 100 lambda 130 AF116272 Homo sapiens T-cell activation protein 532 85 131 W74836 Homo sapiens Human secreted protein 655 100 encoded by gene 108 clone HEBEK93. 132 AF146793 Mus musculus PDCL2 705 58 133 J02459 bacteriophage W (head-tail joining; 68) 336 100 lambda 134 AB011539 Homo sapiens MEGF6 131 58 135 G00827 Homo sapiens Human secreted protein, 523 98 SEQ ID NO: 4908. 136 Y35969 Homo sapiens Extended human secreted 1777 99 protein sequence, SEQ ID NO. 218. 137 AB007861 Homo sapiens KIAA0401 1856 100 138 AK000529 Homo sapiens unnamed protein product 1027 91 139 A17783 unidentified NC28 583 100 140 Y73373 Homo sapiens HTRM clone 921803 561 100 protein sequence. 141 D00762 Homo sapiens proteasome subunit C8 686 93 142 X76091 Homo sapiens DNA binding protein RFX2 3747 99 143 AK000004 Homo sapiens FLJ00004 protein 3106 96 144 Y73377 Homo sapiens HTRM clone 1645941 852 41 protein sequence. 145 Y73377 Homo sapiens HTRM clone 1645941 591 45 protein sequence. 146 U12329 Cricetulus griseus mutant sterol regulatory 373 94 element binding protein-2 147 U80735 Homo sapiens CAGF28 3779 97 148 Y91577 Homo sapiens Human secreted protein 155 96 sequence encoded by gene 2 SEQ ID NO: 250. 149 X00129 Homo sapiens precursor RBP 1005 96 150 X97675 Homo sapiens plakophilin 2a 4288 99 151 J02459 bacteriophage I (tail component; 223) 1111 99 lambda 152 Y94702 Homo sapiens Human androgen shutoff 839 100 gene 3 (AS3) protein sequence. 153 X95325 Homo sapiens DNA-binding protein 1975 100 154 AF171061 Canis familiaris Y-box protein ZONAB-A 1426 89 155 X99584 Homo sapiens SMT3A protein 522 98 156 Y99397 Homo sapiens Human PRO1298 1554 100 (UNQ666) amino acid sequence SEQ ID NO: 210. 157 X78926 Homo sapiens zinc finger protein 426 50 158 J02459 bacteriophage E (capsid component; 341) 1754 100 lambda 159 G02922 Homo sapiens Human secreted protein, 160 48 SEQ ID NO: 7003. 160 AF157317 Homo sapiens AD-015 protein 869 100 161 Y36495 Homo sapiens Fragment of human secreted 111 67 protein encoded by gene 27. 162 Y94870 Homo sapiens Human protein clone 1707 100 HP02545. 163 AF204172 Homo sapiens popeye protein 1 1872 98 164 B01391 Homo sapiens Neuron-associated protein. 376 58 165 Z99259 Schizosaccharomyces putative phosphotransferase 396 43 pombe 166 U28831 Homo sapiens protein that is immuno- 518 55 reactive with anti-PTH polyclonal antibodies 167 AF225419 Homo sapiens HSCARG 1546 100 168 X04412 Homo sapiens plasma gelsolin 4101 100 169 X13482 Homo sapiens U2 snRNP-specific A′ 1284 99 protein (AA 1-255) 170 X59618 Homo sapiens small subunit ribonucleotide 1389 100 reductase 171 AC004882 Homo sapiens similar to cytochrome Bc1 J 326 100 chain; similar to 1BGY (PID: g4139401) 172 AL034374 Homo sapiens dJ483K16.1.1 (novel protein 1651 100 (isoform 1))

TABLE 3 SEQ ID ACCESSION NO: NUMBER DESCRIPTION RESULTS 1 PF01105 emp24/gp25L/p24 family. PF01105B 25.12 1.257e−39 170-222 2 BL00215 Mitochondrial energy BL00215B 10.44 1.900e−09 481-494 transfer proteins. 3 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 6.400e−16 427-440 METAL-BINDI. PD00066 13.92 7.923e−15 371-384 PD00066 13.92 4.600e−14 343-356 PD00066 13.92 3.500e−13 399-412 4 PR00167 CALCIUM CHANNEL PR00167D 13.86 3.400e−34 374-401 SIGNATURE PR00167E 20.54 2.800e−27 1018-1039 PR00167F 16.75 4.000e−20 1551-1566 PR00167G 10.89 5.050e−16 1599-1612 PR00167D 13.86 3.778e−12 1599-1626 6 BL00517 Ribonuclease III family BL00517C 7.29 9.386e−10 98-112 proteins. 8 PR00209 ALPHA/BETA GLIADIN PR00209B 4.88 5.500e−09 325-344 FAMILY SIGNATURE 10 PR00925 NONHISTONE PR00925B 3.73 3.089e−10 11-24 CHROMOSOMAL PROTEIN HMG17 FAMILY SIGNATURE 12 PD01066 PROTEIN ZINC FINGER PD01066 19.43 8.953e−31 27-66 ZINC-FINGER METAL- BINDING NU. 14 PF00521 DNA PF00521G 18.47 7.904e−09 58-92 gyrase/topoisomerase IV, subunit A. 18 BL00107 Protein kinases ATP- BL00107A 18.39 7.207e−11 296-327 binding region proteins. 21 PR00625 DNAJ PROTEIN PR00625A 12.84 2.750e−19 30-50 FAMILY SIGNATURE PR00625B 13.48 4.490e−15 61-82 22 PR00927 ADENINE PR00927B 14.66 6.262e−11 315-337 NUCLEOTIDE PR00927E 14.93 3.160e−09 313-335 TRANSLOCATOR 1 SIGNATURE 26 PR00802 SERUM ALBUMIN PR00802C 12.28 2.370e−11 106-124 FAMILY SIGNATURE PR00802G 14.57 8.734e−09 92-115 28 PR00048 C2H2-TYPE ZINC PR00048A 10.52 5.500e−13 64-78 FINGER SIGNATURE PR00048A 10.52 7.429e−13 149-163 PR00048A 10.52 1.000e−11 35-49 PR00048B 6.02 6.538e−11 137-147 PR00048A 10.52 6.684e−11 121-135 PR00048B 6.02 2.421e−09 80-90 30 PR00988 URIDINE KINASE PR00988A 6.39 8.905e−09 2-20 SIGNATURE 31 BL00315 Dehydrins proteins. BL00315A 9.35 8.119e−09 41-69 33 PR00792 PEPSIN (A1) ASPARTIC PR00792A 11.54 6.250e−23 82-103 PROTEASE FAMILY PR00792D 12.74 5.200e−18 361-377 SIGNATURE PR00792B 12.78 7.750e−14 225-239 PR00792C 9.10 1.000e−12 274-286 34 BL00420 Speract receptor repeat BL00420B 22.67 3.089e−38 408-463 proteins domain proteins. BL00420B 22.67 8.875e−38 618-673 BL00420B 22.67 6.143e−24 272-327 BL00420B 22.67 7.692e−23 167-222 BL00420B 22.67 3.778e−21 62-117 BL00420B 22.67 7.253e−15 515-570 BL00420C 11.90 1.900e−12 493-504 BL00420C 11.90 3.700e−12 703-714 BL00420C 11.90 9.250e−12 40-51 39 BL00667 Respiratory-chain NADH BL00667A 20.80 1.000e−40 22-74 dehydrogenase subunit 1 proteins. 41 BL01160 Kinesin light chain repeat BL01160B 19.54 8.703e−10 407-461 proteins. BL01160B 19.54 2.373e−09 414-468 42 BL00634 Ribosomal protein L30 BL00634 34.38 7.796e−20 100-151 proteins. 50 PD01066 PROTEIN ZINC FINGER PD01066 19.43 9.842e−24 16-55 ZINC-FINGER METAL- BINDING NU. 51 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 8.200e−16 362-375 METAL-BINDI. PD00066 13.92 4.462e−15 334-347 PD00066 13.92 8.615e−15 473-486 PD00066 13.92 5.200e−14 306-319 PD00066 13.92 3.000e−13 390-403 53 BL01160 Kinesin light chain repeat BL01160B 19.54 8.093e−09 586-640 proteins. 54 PF00956 Nuclesosome assembly PF00956B 23.14 2.534e−09 896-937 protein (NAP). 56 BL00479 Phorbol esters/ BL00479A 19.86 6.400e−13 90-113 diacylglycerol binding BL00479B 12.57 5.860e−10 116-132 domain proteins. 57 BL01033 Globins profile. BL01033A 16.94 3.250e−20 26-48 58 PR00910 LUTEOVIRUS ORF6 PR00910A 2.51 4.321e−09 9-22 PROTEIN SIGNATURE 60 BL00518 Zinc finger, C3HC4 type BL00518 12.23 9.143e−10 65-74 (RING finger), proteins. 61 BL00449 ATP synthase a subunit BL00449C 13.17 3.813e−21 32-55 proteins. 62 BL00298 Heat shock hsp90 proteins BL00298A 10.97 1.000e−40 74-119 family proteins. BL00298E 27.30 1.000e−40 321-376 BL00298F 11.21 1.000e−40 409-464 BL00298H 20.50 1.000e−40 553-607 BL00298C 16.40 2.286e−40 186-230 BL00298B 15.64 1.290e−39 134-181 BL00298G 24.57 5.345e−39 465-520 BL00298I 30.07 7.818e−34 661-715 BL00298D 17.97 6.226e−33 242-282 65 BL00223 Annexins repeat proteins BL00223C 24.79 1.000e−40 208-263 domain proteins. BL00223B 28.47 9.679e−39 131-181 BL00223A 15.59 6.000e−27 62-96 BL00223C 24.79 1.000e−16 49-104 BL00223A 15.59 2.306e−16 221-255 66 BL00540 Ferritin iron-binding BL00540A 15.06 1.000e−40 9-50 regions proteins. BL00540B 18.82 1.000e−40 100-155 BL00540C 13.00 7.500e−15 165-177 73 DM00372 CARCINOEMBRYONIC DM00372A 19.18 2.800e−22 19-64 ANTIGEN PRECURSOR DM00372B 20.31 2.523e−19 83-128 AMINO-TERMINAL DOMAIN. 74 PD01168 SYNTHETASE LIGASE PD01168L 9.47 4.500e−09 369-384 PROTEIN ALANYL. 76 BL01033 Globins profile. BL01033A 16.94 7.923e−18 25-47 BL01033B 13.81 1.000e−15 93-105 78 BL01033 Globins profile. BL01033A 16.94 3.250e−20 283-305 BL01033B 13.81 7.000e−14 345-357 79 PR00169 POTASSIUM CHANNEL PR00169A 16.77 2.075e−11 55-75 SIGNATURE 81 PF00992 Troponin. PF00992A 16.67 6.921e−09 417-452 82 PR00935 BAND 4.1 PROTEIN PR00935D 10.20 7.833e−09 240-257 FAMILY SIGNATURE 87 BL00166 Enoyl-CoA BL00166D 22.87 7.250e−20 177-213 hydratase/isomerase BL00166C 18.93 2.537e−19 126-153 proteins. BL00166B 16.92 5.500e−09 80-102 88 BL00166 Enoyl-CoA BL00166D 22.87 7.250e−20 177-213 hydratase/isomerase BL00166C 18.93 2.537e−19 126-153 proteins. BL00166B 16.92 5.500e−09 80-102 89 BL00166 Enoyl-CoA BL00166D 22.87 7.250e−20 146-182 hydratase/isomerase BL00166B 16.92 5.500e−09 80-102 proteins. 91 BL00700 Ribosomal protein L6 BL00700B 18.26 1.000e−40 162-201 proteins 2. BL00700D 21.51 1.000e−40 241-285 BL00700A 19.34 9.308e−33 108-146 BL00700C 11.76 4.000e−13 212-222 95 BL00578 Ribosomal protein S6e BL00578B 15.15 1.000e−40 39-90 proteins. BL00578A 14.24 2.800e−37 1-32 96 BL01020 SAR1 family proteins. BL01020C 15.35 1.000e−40 83-134 BL01020D 19.09 8.071e−40 159-198 BL01020B 11.70 6.750e−38 41-76 BL01020A 11.87 1.400e−28 7-38 97 BL00242 Integrins alpha chain BL00242C 16.86 4.176e−30 319-349 proteins. BL00242E 9.03 3.864e−27 1000-1029 BL00242D 13.57 4.706e−26 391-416 BL00242D 13.57 7.407e−13 455-480 BL00242A 13.80 2.500e−11 92-104 BL00242B 8.13 4.913e−11 222-232 BL00242D 13.57 3.700e−10 324-349 100 BL00227 Tubulin subunits alpha, BL00227C 25.48 2.525e−23 114-166 beta, and gamma proteins. BL00227F 21.16 5.610e−12 400-454 101 BL00227 Tubulin subunits alpha, BL00227C 25.48 2.525e−23 114-166 beta, and gamma proteins. BL00227F 21.16 5.610e−12 298-352 102 BL00162 Eukaryotic-type carbonic BL00162C 17.78 1.000e−19 126-163 anhydrases proteins. BL00162E 14.93 5.922e−16 231-264 BL00162D 15.06 7.864e−12 165-190 BL00162F 22.68 7.750e−11 268-302 BL00162A 22.92 3.282e−10 54-85 104 BL01071 grpE protein. BL01071A 24.88 2.674e−23 76-122 BL01071B 18.21 6.447e−16 191-215 106 PR00002 BONE MATRIX GLA PR00002A 11.56 1.750e−16 481-498 DOMAIN SIGNATURE PR00002B 8.36 7.632e−13 500-511 111 PR00369 FLAVODOXIN PR00369D 20.20 7.261e−16 192-212 SIGNATURE PR00369A 11.29 7.600e−14 84-98 PR00369C 11.27 8.313e−12 168-179 PR00369B 13.29 1.600e−11 137-149 112 PD02784 PROTEIN NUCLEAR PD02784B 26.46 4.713e−16 227-270 RIBONUCLEOPROTEIN. 113 PF00856 SET domain proteins. PF00856B 16.42 3.302e−17 382-404 PF00856A 26.14 3.714e−15 129-166 114 PF00856 SET domain proteins. PF00856B 16.42 3.302e−17 395-417 PF00856A 26.14 3.714e-15 129-166 115 PR00453 VON WILLEBRAND PR00453A 12.79 5.737e−17 1198-1216 FACTOR TYPE A PR00453A 12.79 3.571e−16 439-457 DOMAIN SIGNATURE PR00453A 12.79 5.125e−15 139-157 PR00453A 12.79 1.563e−13 2322-2340 PR00453B 14.65 7.667e−13 178-193 PR00453B 14.65 7.818e−12 1237-1252 PR00453B 14.65 9.455e−12 2362-2377 PR00453B 14.65 1.243e−11 478-493 PR00453C 12.26 4.462e−10 2428-2437 PR00453C 12.26 9.308e−10 544-553 122 DM00668 ZEIN. DM00668C 8.89 8.794e−09 24-48 126 PF00420 NADH- PF00420B 16.51 4.150e−31 61-97 ubiquinone/plastoquinone PF00420A 16.63 3.571e−24 12-43 oxidoreductase chain 4L. 128 BL00405 43 Kd postsynaptic BL00405G 7.78 9.919e−10 285-322 protein. 134 PR00011 TYPE III EGF-LIKE PR00011D 14.03 2.108e−10 18-37 SIGNATURE PR00011B 13.08 6.055e−09 18-37 PR00011A 14.06 6.178e−09 18-37 139 BL00472 Small cytokines BL00472C 20.76 6.850e−34 65-102 (intercrine/chemokine) C- BL00472B 14.67 8.579e−15 44-62 C subfamily signatur. BL00472A 7.45 2.895e−11 11-23 140 PR00988 URIDINE KINASE PR00988A 6.39 8.905e−09 2-20 SIGNATURE 141 BL00388 Proteasome A-type BL00388A 23.14 5.500e−39 8-54 subunits proteins. BL00388B 31.38 1.310e−27 66-108 BL00388C 18.79 3.842e−10 121-143 142 PD02699 PROTEIN DNA- PD02699C 24.84 1.000e−40 614-661 BINDING BINDING PD02699A 8.91 3.250e−35 235-264 DNA. PD02699B 18.28 6.571e−21 500-524 143 DM01970 0 kw ZK632.12 YDR313C DM01970B 8.60 2.612e−13 551-564 ENDOSOMAL III. 144 PR00624 HISTONE H5 PR00624G 4.08 6.900e−09 180-200 SIGNATURE 146 BL00038 Myc-type, ‘helix-loop- BL00038B 16.97 9.027e−10 57-78 helix’ dimerization domain proteins. 147 PR00209 ALPHA/BETA GLIADIN PR00209B 4.88 4.494e−12 393-412 FAMILY SIGNATURE 149 PR00179 LIPOCALIN PR00179B 9.56 2.895e−13 128-141 SIGNATURE PR00179A 13.78 3.250e−11 40-53 PR00179C 19.02 6.040e−11 158-174 152 PR00929 AT-HOOK-LIKE PR00929C 5.26 8.759e−09 55-66 DOMAIN SIGNATURE 153 BL00352 ‘Cold-shock’ DNA-binding BL00352B 23.66 5.886e−25 119-158 domain proteins. BL00352A 12.19 1.429e−17 93-108 154 BL00352 ‘Cold-shock’ DNA-binding BL00352B 23.66 5.886e−25 119-158 domain proteins. BL00352A 12.19 1.429e−17 93-108 155 BL00299 Ubiquitin domain proteins. BL00299 28.84 2.250e−25 32-84 156 PF00534 Glycosyl transferases PF00534B 14.47 8.364e−15 325-349 group 1. 157 BL00028 Zinc finger, C2H2 type, BL00028 16.07 8.615e−11 46-63 domain proteins. 164 BL00880 Acyl-CoA-binding protein. BL00880 17.52 8.875e−34 46-96 168 BL00740 MAM domain proteins. BL00740B 19.76 3.813e−09 690-711 170 BL00368 Ribonucleotide reductase BL00368A 36.98 1.000e−40 84-139 small subunit proteins. BL00368B 22.06 5.846e−26 161-187 172 BL01188 GNS1/SUR4 family BL01188C 22.65 6.667e−10 154-205 proteins.

TABLE 4 SEQ ID PFAM NO: PFAM NAME DESCRIPTION p-value SCORE 1 EMP24_GP25L emp24/gp25L/p24 family 6.4e−90 312.2 2 mito_carr Mitochondrial carrier proteins 1.1e−09 36.2 3 SCAN SCAN domain   1e−56 201.9 4 ion_trans Ion transport protein 5.5e−260 877.1 6 Skp1 Skp1 family 2.2e−64 227.3 12 zf-C2H2 Zinc finger, C2H2 type 3.2e−53 190.2 13 Rap_GAP Rap/ran-GAP 5.4e−117 402.1 17 MSP_domain MSP (Major sperm protein) domain 2.9e−10 47.5 18 pkinase Eukaryotic protein kinase domain 0.022 12.5 21 DnaJ DnaJ domain 2.4e−34 127.5 22 mito_carr Mitochondrial carrier proteins 1.1e−85 296.7 24 PH PH domain 2.4e−14 56.3 26 transport_prot Serum albumin family 2.4e−06 11.0 28 zf-C2H2 Zinc finger, C2H2 type 2.7e−22 87.5 31 zf-A20 A20-like zinc finger 0.0056 23.3 32 Dihydrooratase Dihydroorotase-like 7.6e−88 305.3 33 asp Eukaryotic aspartyl protease 5.7e−211 714.3 34 SRCR Scavenger receptor cysteine-rich 4.3e−187 634.9 domain 37 F-box F-box domain. 0.089 19.3 39 NADHdh NADH dehydrogenases 1.3e−39 145.0 42 Ribosomal_L30 Ribosomal protein L30p/L7e 1.2e−10 40.0 43 G_Adapt_CT Gamma-adaptin, C-terminus 1.7e−67 237.6 50 KRAB KRAB box 7.4e−12 52.8 51 SCAN SCAN domain 5.4e−67 236.0 55 PH PH domain 1.1e−06 28.9 56 SH3 SH3 domain 1.8e−14 61.5 57 globin Globin 2.1e−26 96.5 60 NHL NHL repeat 1.7e−46 167.9 61 ATP-synt_A ATP synthase A chain 1.3e−10 48.7 62 HSP90 Hsp90 protein 0 1548.4 65 annexin Annexin 1.9e−77 270.7 66 ferritin Ferritins 4.1e−114 386.1 67 PUA PUA domain 1.7e−16 68.2 69 DUF34 Domain of unknown function DUF34 1.2e−33 125.2 73 ig Immunoglobulin domain 0.0015 16.5 75 Exonuclease Exonuclease 0.076 −29.3 76 globin Globin 5.9e−53 189.1 78 globin Globin 4.6e−57 203.0 79 K_tetra K+ channel tetramerisation domain 5.6e−22 86.4 81 PH PH domain 2.2e−21 81.4 82 Band_41 FERM domain (Band 4.1 family) 1.1e−22 73.0 85 oxidored_q1 NADH-Ubiquinone/plastoquinone 3.6e−06 25.3 (complex I) 87 ECH Enoyl-CoA hydratase/isomerase family 4.1e−52 186.6 88 ECH Enoyl-CoA hydratase/isomerase family 4.1e−52 186.6 89 ECH Enoyl-CoA hydratase/isomerase family 1.3e−20 81.9 91 Ribosomal_L6 Ribosomal protein L6   9e−99 341.6 95 Ribosomal_S6e Ribosomal protein S6e 1.8e−90 314.0 96 arf ADP-ribosylation factor family 5.4e−98 339.0 97 FG-GAP FG-GAP repeat 1.4e−77 271.2 100 tubulin Tubulin/FtsZ family 1.8e−49 177.8 101 tubulin Tubulin/FtsZ family 1.8e−49 177.8 102 carb_anhydrase Eukaryotic-type carbonic anhydrase 2.3e−48 174.1 104 GrpE GrpE 5.9e−70 245.8 111 FAD_binding FAD binding domain 7.2e−122 418.3 112 rrm RNA recognition motif. 9.7e−12 52.4 113 SET SET domain 3.3e−34 127.1 114 SET SET domain 3.3e−34 127.1 115 fn3 Fibronectin type III domain 2.6e−292 984.5 126 oxidored_q2 NADH-ubiquinone/plastoquinone 1.8e−44 161.2 oxidoreduct 132 Phosducin Phosducin 0.097 9.7 139 IL8 Small cytokines (intecrine/chemokine), 3.6e−38 131.6 inter 141 proteasome Proteasome A-type and B-type 6.1e−39 142.8 142 RFX_DNA_binding RFX DNA-binding domain 4.1e−43 156.7 143 RhoGEF RhoGEF domain 8.1e−64 225.4 146 HLH Helix-loop-helix DNA-binding domain 1.4e−05 31.9 147 BRCT BRCA1 C Terminus (BRCT) domain 1.2e−28 108.6 149 lipocalin Lipocalin/cytosolic fatty-acid binding 2.5e−41 146.4 pr 150 Armadillo_seg Armadillo/beta-catenin-like repeats 7.8e−10 46.1 153 CSD ‘Cold-shock’ DNA-binding domain 1.1e−33 124.6 154 CSD ‘Cold-shock’ DNA-binding domain 1.1e−33 124.6 156 Glycos_transf_1 Glycosyl transferases group 1 6.7e−39 142.7 157 zf-C2H2 Zinc finger, C2H2 type 0.00041 27.1 162 ig Immunoglobulin domain   1e−06 26.7 164 ACBP Acyl CoA binding protein 1.1e−39 145.2 165 DUF60 Domain of unknown function DUF60 2.2e−36 134.3 168 Gelsolin Gelsolin repeat. 9.3e−109 374.7 169 LRR Leucine Rich Repeat 0.00025 27.8 170 ribonuc_red Ribonucleotide reductases 1.5e−122 358.6 172 GNS1_SUR4 GNS1/SUR4 family 2.7e−09 −26.6

TABLE 5 SEQ ID NO: of full- SEQ ID NO: SEQ ID NO: SEQ ID NO: Priority docket length of full-length of contig of contig number_corresponding nucleotide peptide nucleotide peptide SEQ ID NO: in priority SEQ ID NO: in sequence sequence sequence sequence application USSN 09/515,126 1 173 345 517 788CIP2_3 788_9798 2 174 346 518 788CIP2_5 788_10429 3 175 347 519 788CIP2_6 788_10672 4 176 348 520 788CIP2_7 788_10828 5 177 349 521 788CIP2_8 788_10879 6 178 350 522 788CIP2_9 788_11026 7 179 351 523 788CIP2_10 788_11045 8 180 352 524 788CIP2_11 788_11381 9 181 353 525 788CIP2_12 788_11481 10 182 354 526 788CIP2_13 788_11927 11 183 355 527 788CIP2_14 788_12700 12 184 356 528 788CIP2_15 788_12706 13 185 357 529 788CIP2_16 788_12741 14 186 358 530 788CIP2_17 788_12744 15 187 359 531 788CIP2_18 788_13017 16 188 360 532 788CIP2_20 788_13633 17 189 361 533 788CIP2_21 788_13637 18 190 362 534 788CIP2_22 788_13645 19 191 363 535 788CIP2_23 788_13695 20 192 364 536 788CIP2_24 788_13756 21 193 365 537 788CIP2_25 788_13858 22 194 366 538 788CIP2_26 788_13886 23 195 367 539 788CIP2_27 788_14039 24 196 368 540 788CIP2B_2 788_31 25 197 369 541 788CIP2B_3 788_54 26 198 370 542 788CIP2B_4 788_58 27 199 371 543 788CIP2B_5 788_768 28 200 372 544 788CIP2B_6 788_1718 29 201 373 545 788CIP2B_7 788_1748 30 202 374 546 788CIP2B_8 788_1751 31 203 375 547 788CIP2B_9 788_1850 32 204 376 548 788CIP2B_10 788_1946 33 205 377 549 788CIP2B_11 788_1967 34 206 378 550 788CIP2B_12 788_1994 35 207 379 551 788CIP2B_13 788_2331 36 208 380 552 788CIP2B_14 788_2725 37 209 381 553 788CIP2B_15 788_2735 38 210 382 554 788CIP2B_16 788_2961 39 211 383 555 788CIP2B_18 788_3039 40 212 384 556 788CIP2B_19 788_3287 41 213 385 557 788CIP2B_20 788_4305 42 214 386 558 788CIP2B_21 788_4356 43 215 387 559 788CIP2B_22 788_4389 44 216 388 560 788CIP2B_24 788_4458 45 217 389 561 788CIP2B_25 788_4685 46 218 390 562 788CIP2B_26 788_4825 47 219 391 563 788CIP2B_27 788_4829 48 220 392 564 788CIP2B_28 788_4856 49 221 393 565 788CIP2B_29 788_4917 50 222 394 566 788CIP2B_30 788_4939 51 223 395 567 788CIP2B_31 788_4943 52 224 396 568 788CIP2B_32 788_5175 53 225 397 569 788CIP2B_33 788_5254 54 226 398 570 788CIP2B_34 788_5566 55 227 399 571 788CIP2B_35 788_5725 56 228 400 572 788CIP2B_36 788_5794 57 229 401 573 788CIP2B_37 788_5796 58 230 402 574 788CIP2B_38 788_5841 59 231 403 575 788CIP2B_39 788_5922 60 232 404 576 788CIP2B_40 788_5942 61 233 405 577 788CIP2B_41 788_5980 62 234 406 578 788CIP2B_42 788_6025 63 235 407 579 788CIP2B_43 788_6151 64 236 408 580 788CIP2B_44 788_6849 65 237 409 581 788CIP2B_45 788_7191 66 238 410 582 788CIP2B_46 788_7281 67 239 411 583 788CIP2B_47 788_8402 68 240 412 584 788CIP2B_48 788_9636 69 241 413 585 788CIP2B_49 788_9645 70 242 414 586 788CIP2B_50 788_9784 71 243 415 587 788CIP2B_51 788_10075 72 244 416 588 788CIP2B_52 788_10223 73 245 417 589 788CIP2B_53 788_10369 74 246 418 590 788CIP2B_54 788_10413 75 247 419 591 788CIP2B_55 788_10441 76 248 420 592 788CIP2B_56 788_10607 77 249 421 593 788CIP2B_57 788_10625 78 250 422 594 788CIP2B_58 788_10857 79 251 423 595 788CIP2B_59 788_10868 80 252 424 596 788CIP2B_60 788_10962 81 253 425 597 788CIP2B_61 788_11218 82 254 426 598 788CIP2B_62 788_11224 83 255 427 599 788CIP2B_63 788_11251 84 256 428 600 788CIP2B_64 788_11262 85 257 429 601 788CIP2B_65 788_11597 86 258 430 602 788CIP2B_66 788_11708 87 259 431 603 788CIP2B_67 788_11719 88 260 432 604 788CIP2B_68 788_11719 89 261 433 605 788CIP2B_69 788_11719 90 262 434 606 788CIP2B_70 788_11823 91 263 435 607 788CIP2B_71 788_11865 92 264 436 608 788CIP2B_72 788_11939 93 265 437 609 788CIP2B_73 788_11961 94 266 438 610 788CIP2B_74 788_12119 95 267 439 611 788CIP2B_75 788_12128 96 268 440 612 788CIP2B_76 788_12190 97 269 441 613 788CIP2B_77 788_12212 98 270 442 614 788CIP2B_78 788_12233 99 271 443 615 788CIP2B_79 788_12392 100 272 444 616 788CIP2B_80 788_12518 101 273 445 617 788CIP2B_81 788_12518 102 274 446 618 788CIP2B_82 788_12525 103 275 447 619 788CIP2B_83 788_12594 104 276 448 620 788CIP2B_84 788_12737 105 277 449 621 788CIP2B_85 788_12818 106 278 450 622 788CIP2B_86 788_12825 107 279 451 623 788CIP2B_87 788_12868 108 280 452 624 788CIP2B_88 788_13029 109 281 453 625 788CIP2B_89 788_13031 110 282 454 626 788CIP2B_90 788_13037 111 283 455 627 788CIP2B_91 788_13051 112 284 456 628 788CIP2B_92 788_13061 113 285 457 629 788CIP2B_93 788_13105 114 286 458 630 788CIP2B_94 788_13105 115 287 459 631 788CIP2B_95 788_13131 116 288 460 632 788CIP2B_96 788_13281 117 289 461 633 788CIP2B_97 788_13325 118 290 462 634 788CIP2B_98 788_13453 119 291 463 635 788CIP2B_99 788_13557 120 292 464 636 788CIP2B_100 788_13638 121 293 465 637 788CIP2B_101 788_13638 122 294 466 638 788CIP2B_102 788_13655 123 295 467 639 788CIP2B_103 788 _13656 124 296 468 640 788CIP2B_104 788_13668 125 297 469 641 788CIP2B_105 788_13681 126 298 470 642 788CIP2B_106 788_13725 127 299 471 643 788CIP2B_107 788_13796 128 300 472 644 788CIP2B_108 788_13802 129 301 473 645 788CIP2B_109 788_13814 130 302 474 646 788CIP2B_110 788_13816 131 303 475 647 788CIP2B_111 788_13836 132 304 476 648 788CIP2B_112 788_13852 133 305 477 649 788CIP2B_113 788_13855 134 306 478 650 788CIP2B_114 788_13990 135 307 479 651 788CIP2B_115 788_14056 136 308 480 652 788CIP2B_116 788_14066 137 309 481 653 788CIP2B_117 788_14070 138 310 482 654 788CIP2C_1 788_360 139 311 483 655 788CIP2C_2 788_1175 140 312 484 656 788CIP2C_3 788_3964 141 313 485 657 788CIP2C_4 788_5522 142 314 486 658 788CIP2C_5 788_5537 143 315 487 659 788CIP2C_6 788_5573 144 316 488 660 788CIP2C_7 788_5868 145 317 489 661 788CIP2C_8 788_5868 146 318 490 662 788CIP2C_9 788_6265 147 319 491 663 788CIP2C_10 788_7013 148 320 492 664 788CIP2C_11 788_7017 149 321 493 665 788CIP2C_12 788_7019 150 322 494 666 788CIP2C_13 788_8583 151 323 495 667 788CIP2C_14 788_8792 152 324 496 668 788CIP2C_15 788_8816 153 325 497 669 788CIP2C_16 788_8916 154 326 498 670 788CIP2C_17 788_8916 155 327 499 671 788CIP2C_18 788_8950 156 328 500 672 788CIP2C_19 788_9067 157 329 501 673 788CIP2C_20 788_9254 158 330 502 674 788CIP2C_21 788_9285 159 331 503 675 788CIP2C_22 788_10049 160 332 504 676 788CIP2C_23 788_10858 161 333 505 677 788CIP2C_24 788_11054 162 334 506 678 788CIP2C_25 788_11872 163 335 507 679 788CIP2C_26 788_12230 164 336 508 680 788CIP2C_27 788_12369 165 337 509 681 788CIP2C_28 788_12464 166 338 510 682 788CIP2C_29 788_12708 167 339 511 683 788CIP2C_30 788_13024 168 340 512 684 788CIP2C_31 788_13199 169 341 513 685 788CIP2C_32 788_13601 170 342 514 686 788CIP2C_33 788_13666 171 343 515 687 788CIP2C_34 788_13749 172 344 516 688 788CIP2C_35 788_14042

TABLE 6 Predicted Amino acid sequence (A = Alanine C = Cysteine, beginning Predicted end D = Aspartic Acid, E = Glutamic Acid, nucleotide nucleotide F = Phenylalanine, G = Glycine, H = Histidine, location location I = Isoleucine, K = Lysine, L = Leucine, M = Methionine, corresponding corresponding N = Asparagine, P = Proline, Q = Glutamine, to first amino to last amino R = Arginine, S = Serine, T = Threonine, V = Valine, SEQ acid residue of acid residue of W = Tryptophan, Y = Tyrosine, X = Unknown, * = Stop ID peptide peptide codon, / = possible nucleotide deletion, \ = possible NO: Method sequence sequence nucleotide insertion 517 A 40 359 LKIPMQFLHSGFWFSFFVFFGF*KFGFGP QGGRQGG\NKTKGEKLPPGKKKIPGPNP QENREKKGPPKTLKKFGNLKKKGGKTR GPRGEKNSDPKGTPGQNPGNKKK 518 A 50 371 VVVAPVVVGSGRA/PRHPAPAAMHSRRP DGFDGLGYRGGARDEQGFGGAFPARSFI TGSDLGHWVTTLTDI\PGRRN\LHWGVK SPPYGVPTNCTPYEGPTEDPFDSGSG 519 A 114 380 DGFPLSFYNTPKSPTSEILFPLSEILFSGPF GWLAAPSCFGFSCHFLKKGFLGPGTVAH SCNPSTLGGRGGWI/T/RGQEIKTILANTV KP 520 A 13 411 GPLRVDTWSNVESLACAQREEEEENER KKLPGTASPEKKQE\LVDEPAVGESKEE KIELKSITADGESPPASKINMDALQPNEN EDKSPYPTPESTGEEDEEEPEMPVGPRPR PLSELHLKEKTVPMPEASAC 521 A 1 451 KINTELQTEVAMLKSMVLWLGEQVQSL QLQQQLRHHFNHIHICVTNSEYNQSEYP WDLVKAHLQGAFTSNITFDTGEFQNTIID STK*PHDL*ASLDCS*I*DGLSILASVLM LAAPVLALQTPSWMLFMALPVSLSRLK AYFLVSVLS 522 A 354 549 IFWVGKNALHEDE*NTERRTDDIPVWDH EFLKVDQGTLFELILAANYLYIRGWLDV TCKTGANMI 523 A 448 489 YARSAIGLLVFQSLMLVAAGDQFLFRP* GE*RQPR**LKMTKHPPNRRGISFEVGAQ LEARDRLKNWYPAHIEDIDYEEGKVLIH FKRWNHRYDEWFCWDSPYLRPLEKIQL RKEGLHEEDGSSVSNKSGKFCSMQGQPL GCLCFNL 524 A 35 402 KLFLGFPILHGSSSPP*LKVPSLTHKVSAL VSSNSF*VMYLVIFHMNMKVAWRRGSL LHSFQLGFYKSQHFGRPRWADRLRLGV RDQPGQHGETPSLLVQKKI 525 A 203 3 GPKPFSLGHKGKSCVKKNIFFFFFGGSHL *MQLLRRLRWEDQLSSGGRGCSGP*LHH CTVAWATEQD 526 A 617 14 VFTGNYNSKMRIWVGTQPNHISAHPHW VMVISVSSRNALIDMPRNPCLTSCVCLSI QSSRRLRLTIRIFMPDSWLKSCLTSYVCL SIQSSRCLRLTIRIFMPDSWLKSCLTSYVC LSIQSSRRLQLTIRIFMPDSWLKSCLTSYV CLSIQSSRCLQLTIRIFMPDSWLKSCLTSY VCLLIQSSRCLKLTVRIFMPLVPRLA 527 A 149 759 SRMTKKRKRQHDFQKVKLKVGKKKPK LQNATPTNFKTKTIHLPEQLKEDGTLPTN NRKLNIKDLLSQMHHYNAGVKQRALLG LKDLLSQYPFIIDAHLLNILSEVTAVFTD KDANVRLAAVQLLQFLAPKIRAEQISPFF PLVSAHLSSAMTHITEGIQEDSLKVLDIL LEQYPALITGRSSILLKNFVELISHQQLSK GLDK 528 A 117 369 WLPGEMAVEPPADRLQEPLTFRDGAED FTQEEWGQLDPTQRILYRDVMLETFDHL LSIGPELTKPEDISQLEQGTDLWVALETLT 529 A 836 433 AVGGSSRSHGYRRPAGTRPVSHAQDPSA PPPAS/RLPCIEARGGQ*SLPSLILQPGGA GWGILLPNSVLPPAPCTPQSHVSLPSAGP CAPLGGRRQAPLGPRAAEPCRVRGCGA AALSCALPALGRGTFGWKTGI 530 A 1282 210 VRCTLSPACQTPPQGPHRSSGPVLSSPCP APVGEWLPPNGP*SDDELPLSQQCKCRT KSLAPPSSA*GS*HRT*LPGTGH*CGLARI ESSAGVLEEASAAK*GSGEAPGSKQHSS SPAATSAPSAGSWHPGTAAAHPPG/SSAS FHTFAAFTVSLSASELGS**QSRVKHFFL SCSSTSCCSRALIHTKSSESGMSSRRSAFS SPSSGGISLSWLGPKSRISSVTSSRCSRVS *NFLSSEVTFVS\VSSHSSPQATLE*LCVK SQTSTMLSYAVELPSMPEPAPLLG*GLSH SHPHAVRDSGEAC*SMGSVTGPLYSGYK EEVVCCTLVEVFPSLLQVSRNPRMPFDF LGILICTPWGS 531 A 12 245 CAKNECQGGQVESGSF*AIYL*TVI\YLC VYLSVCLSIYLSVYLSIYLSIYLSIYLSIYP SIHPSIQLASI*SIFLWG 532 A 1 849 RKMAGSPELVVLDPPWDKELAAGTESQ ALVSATPREDFRVRCTSKRAVTEMLQLC GRFVQKLGDALPEEIREPALRDAQWTFE SAVQENISINGQAWQEASDNCFMDSDIK VLEDQFDEIIVDIATKRKQYPRKILECVIK TIKAKQEILKQYHPVVHPLDLKYDPDPA PHMENLKCRGETVAKEISEAMKSLPALI EQGEGFSQVLRMQPVIHLQRIHQEVFSS CHPDAPENFITQIETTPTETASRKTS DVVLKRKQTKDCPQRKWYPLRPKKINL DT 533 A 120 804 DCKNRNCSKGDSVPMHQQKRQPELVEG NLPVFVFPTELIFYADDQSTHKQVLTLY NPYEFALKFKVLCTTPNKYVVVDAAGA VKPQCCVDIVIRHRDVRSCHYGVIDKFR LQVSEQSQRKALGRKEVVATLLPSAKEQ QKEEEEKRLKEHLTESLFFEQSFQPENRA VSSGPSLLTVFLGVVCIAALMLPTLGDV ESLVPLYLHLSVNQKLVAAYILGLITMAI LRT 534 A 479 1906 SMISFCPDCGKSIQAAFKFCPYCGNSLPV EEHVGSQTFVNPHVSSFQGSKRGLNSSF ETSPKKVKWSSTVTSPRLSLFSDGDSSES EDTLSSSERSKGSGSRPPTPKSSPQKTRK SPQVTRGSPQKTSCSPQKTRQSPQTLKRS RVTTSLEALPTGTVLTDKSGRQWKLKSF QTRDNQGILYEAAPTSTLTCDSGPQKQK FSLKLDAKDGRLFNEQNFFQRAAKPLQV NKWKKLYSTPLLAIPTCMGFGVHQDKY RFLVLPSLGRSLQSALDVSPKHVLSERSV LQVACRLLDALEFLHENEYVHGNVTAE NIFVDPEDQSQVTLAGYGFAFRYCPSGK HVAYVEGSRSPHEGDLEFISMDLHKGCG PSRRSDLQSLGYCMLKWLYGFLPWTNC LPNTEDIMKQKQKFVDKPGPFVGPCGH WIRPSETLQKYLKVVMALTYEEKPPYA MLRNNLEALLQDLRVSPYDPIGLPMVP 535 A 3828 2909 AECEEVRRKSELFNPVSLDCKLRQKAIA EVDVGTDKAQNSDPILDTSSLVPGCSSV DNIKSSQTSQNQGLGRPTLEGDEETSEVE YTVNKGPASSNRDRVPPSSEASEHHPRH RVSSQAEDTSSSFDNLFIDRLQRITIADQ GEQQSEENASTKNLTGLSSGTEKKPHYM EVLEMRAKNPVPQLRKFKTNVLPFRQN DSSSHCQKSGSPISSEERRRRDKQHLDDI TAARLLPLHHMPTQLLSIEESLALQKQQ KQNYEEMQAKLAAQKLAERLNIKMRSY NPEGESSGRYREVRDEDDDWSSDEF 536 A 45 257 TLEFESHIIFMSHRIFFFCYLKAKIKILGW ARWLVPVIPALWEAEVGRSFEARSSRTA WATYGGSLFYKK 537 A 1071 472 KMACNIPNQRQRTLSTTGEALYEILGLH KGASNEEIKKTYRLKLALKHHPDKNPDDP AATEKFKEITDAHAILTDISKRSIYDKYG SLGLYVAEQFGDENVNTYFMLSSWWA KALFVIVGLLTGCYFCCCLCCCCNCCCG HCRPESSVPEEDFYVSPEDLEEQIKSDME KDVDFPVFLQPTNANEKTQLIKEGSRSY CTDS 538 A 1 1528 RLAAAGADPAGSRGGRGSREAPTEAAR CRASPPPRAGAMRGSPGDAERRQRWGR LFEELDSNKDGRVDVHELRQGLARLGG GNPDPGAQQGISSEGDADPDGGLDLEEF SRYLQEREQRLLLMFHSLDRNQDGHIDV SEIQQSFRALGISISLEQAEKILHSMDRDG TMTIDWQEWRDHFLLHSLENVEDVLYF WKHSTVLDIGECLTVPDEFSKQEKLTGM WWKQLVAGAVAGAVSRTGTAPLDRLK VFMQVHASKTNRLNILGGLRSMVLEGGI RSLWRGNGINVLKIAPESAIIKFMAYEQIK RAILGQQETLHVQERFVAGSLAGATAQT IIYPMEVLKTRLTLRRTGQYKGLLDCAR RILEREGPRAFYRGYLPNVLGIIPYAGIDL AVYETLKNWWLQQYSHDSADPGILVLL ACGTISSTCGQIASYPLALVRTRMQAQA SIEGGPQLSMLGLLRHILSQEGMRGLYR GIAPNFMKVIPAVSISYVVYENMKQALG VTSRSFC 539 A 474 1098 RRRCFQGFLKKKKIIQPKGEGTGRGRGV VPFIHNIVNIHGLYYMCFSLVFACLSVRL SVWGLDLRSRAPLLPQPPPPCPTLGTELE QVWPPNHRWEPAGSAWATASTLAPESC VCKCVCVYACLSMCMYVHGEPVCKYV QRETVTVTEAGGRWGCWRRGLPGADSS QVDPPQVSGRCDRVALRAELLGTQGPQ PALLAVHVPTLDGR 540 A 1178 1 VSFLLWTSGNKEDGRNGPAVPSRHTGG SRTWRMPK*QEGENPCPLPKRNLGNA*T FNS*KRTGPG/SPDSKGPDLGPI/SVPHILS GSPQPSS\PTLPQLHDLLAIPVMQGREAG GLRGHRALPLA*EEAGRALQLPLSQPPP CRPAAPFFNQ/GPQGCCPWRGSGHSPGP QLGG*EPGAYSGWQQTSRMSGVAKPGS GVRPATGSPTRVPPGACSQSLPGTAPSPR PPGPPALLRPAGVGALQGHQSRWQRQT PRAREPRPVPLCSLPAPPGSVGPGPRDSQ RRIAGCP*GQTSIFGQCHWGSVGSLGPPR CAGSPCGPLGSR*VHSGLHSSRSASWLP REQQGS/PGGGQGPLVWPHSSALRPPGN PGNLPPPIAPKPKRAIPARDPAPPPGLQH E 541 A 1 438 MLAIFKYLINNRLITT*Q/WTEKLTS*QMI TIHNTKGLT*SLILLSLIIFIATTNLLGLLP YSFTPTTQLSINLSMAIPL*TVAEFIGFRS KIKNALSHFLPQGTPTPLIPILVIIETISLLI QPILLDVLLTANITAGHLLIHLN 542 A 13 577 LNINKKYNYIKR*DYYKIII\NKKMNII*IIN K*YNKE*YK*NMKKIIK\Y*IYYSQVSTPT IVKVSRNLGKVGSKCCKHPEAKRMPCA EDYLYVVLNQL*VLHEKTPVNDRVTKC *KESLVNRRPCFSTLEVDKTYVTKKFNA ETFTFHADICTLSEKERQIKKQTALDELV KHKPKATKEQLKAVMDD 543 A 297 0 SSSSSSPSTPGPPSPFLRQPPFGLNPFLPAG D*GTQRGGSGRGGRGRGR/DGSTNEGRG ERRGQG 544 A 395 1 AKRKSVPEEIWKSRGQFKNQQLNKENN LGQEIATCTKIPSRKRDIEFNEFVKNFTV RSILVAEQIDPMEENCHKYGTC*KMLKQ NSDLIIQ\ESMMEKKKPCKYSECGRTFRG HITLVQHQITHCGERPCI 545 A 291 209 NNKNKIASKRKKNSPEPGGLTTEL/YQTS KEELEPILLNLFQKIEKEEILSNSFRETSIA LIPKPGKDITKEVNHRPISLMNRDAKVL NKMLSN*IQ 546 A 419 3 GKPPFYFGGPGFFPPPV/YFKPPPPKNFFG PPKKKKFPPPPGVYFFFFLRPPPPLFFFFF FFFFFFFF*EGGGEKPSPP/IKKGFFKPKKF FSPPPGGKKKPPFLKKKKKKKDVPQSSN LESSLSFPIHKTGTIIPASLNV 547 A 28 414 HTLLLQCLSAVAFSQPCPHPSRL/CNPCC ASGPPCSS*ACCPRFCQSCPVC*PQNLLS SCPSPDLALTSPSAGRPRCWWNCCQLTP CLSLQTQEWTGAHLQLRRWGTQAGPEE PVHQRTVGSTSHSCSYA 548 A 419 2 RWSARSRITGEEGSESAVQER*LTYHTH GGIANRDRTHCPVSLVN\VSSMSAGDVIT AGKMQGKVVLAEPTAALCHGN\GLHYY RQDWSHAAAYVTVPPVRVDTNTSTYLM SLLANDTLNIVASDRRPFTTKQKAMGNV 549 A 201 550 GCLVSLGVSHAWQRVGTWEIFVG*MNA G*MSA*TRGTEISRIGQWMGGEGMSGLT SWFLLGEMNP*GLRELNLLLTLSITMNG ETIACAEGCQAICDTGTSLLT/GPTSPIAQ HQSDI 550 A 1 526 LLDNCQMTVLGAPTCIHGNTVSVICTGS LTQPLFPCLANVSDPYLSAVPEGSALICL EDKRLRLVDGDSRCAGRVEIYHDGFWG TICDDGWDLSDAHVVCQKLSCGVAFNA TVSAHFG/VGAGPIW/LDDL/NCTGMESH LRQ/CPF 551 A 369 130 FFFFFFXXXGVXGLNFFFFXFFXXFFFFFF FXFXXXXXFFFFFXGXXXXFXXFXXXX XFFFFFFFFFFFFFFFFFFFXCY 552 A 255 338 EVMHFCLFVF*DRVLLCHPGWSAVAQS Q 553 A 120 383 TAPKKKKGGAPLGGPPRGPILPRIPENRR PP**GALLKPSPGPRFNTAWPGKPAPRES LKRAPSLGGGPKRPNPPPEVKASGADKK L 554 A 3 365 YHIVQPSP*PLAGALSALLMTSGLAMRS HFHSITLLILGLLSSTLTIYQ*WRDVTRES TYQGHHTPPVRKGLRYGIILFITSEVIFFA GLF*AFYHSSLAPTPQLGGHWPPTGITPL NTLE 555 A 1 125 LMNSNSIPPIPLRPTHTPPMKKL*IRTAYP RFRYDQLIHLL*KNFLPLTLALLI*HVKK KKKK 556 A 372 86 PACGGFSPPPPPKFFFFPPPLFFFGGGGPP FPPPPKGFFFPNPPRGFFFPPP*KKKFFFPP PPFFCPPPVFFFCPPPLFFFFFFFFFFFFFFF FF 557 A 2 376 FVAAEVRDGGGGAGTRPPSGRMKRRNA DCSKLRRPLKRNRITEGIYGSTFLYLKFL VVWALVLLADFVLEFRFEYLWPFWLFIR SVYDSFRYQGLVCLVIHVFHVWGPFSCL E*MLLAILR*LC 558 A 39 459 VLNHSYMIPGRRNVTRCISRQLEVQPHA LELPDKCSLAFVVRIKRIDGGSLLVQRTI ARLCLKKIFSGVFLKMLRIVEPYVT*GFP NLKSVWEFILKHGQAKVKNKTIGQTQW LTPVIPALWEAKDGWIMRSGDRDHPGQ 559 A 2 456 EVRSHVKVLGEMLVMYRRPGQAPPDQE ALQVVYERCEKLRPTLFRLASDTTDDDD ALSEILQANDLLTQGVLLYKQVMEGRV TFGNRVTSSLGDIPVSRVFQNPAGCMKT CPLIDLEVDNGPAQMGTVVPSLLHQDLA ALGNSDAPVTGMV 560 A 54 1063 RRRADGCIYGVSRRARVVAYRRDEMWS EGRYEYERIPRERAPPRSHPSDESGYRW TRDDHSASRQPEYRDMRDGFRRKSFYSS HYARERSPYKRDNTFFRESPVGRKDSPH SRSGSSVSSRSYSPERSKSYSFHQSQHRK SVRPGASYKRQNEGNPERDKERPVQSLK TSRDTSPSSGSAVSSSKVLDKPSRLTEKE LAEAASKWAAEKLEKSDESNLPEISEYE AGSTAPLFTDQPEEPESNTTHGIELFEDS QLTTRSKAIASKTKEIEQVYRQDCETFG MVVKMLIEKDPSLEKSIQFALRQNLHEI GERCVEELKHFIAEYDTSTQDFGEPF 561 A 1887 442 LLIREVFIQVLIKLRVCFVQCFSEADRDI MTLANHWNCPVLSSDSDFCIFDLKTGFC PLNSFQWRNMNTIKGTQNYIPAKCFSLD AFCHHFSNMNKALLPLFAGLCGNDHVN LPIMETFLSKARLPLGATSSKGRRHHRIL GLLNWLSHFANPTEALDNVLKYLPKKD RENVKELLCCSMEEYQQSQVKLQDFFQ CGTYVCPDALNLGLPEWVLVALAKGQL SPFISDALVLRRTILPTQVENMQQPNAHR ISQPIRQIIYGLLLNASPHLDKTSWNALPP QPLAFSEVERINKNIRTSIIDAVELAKDHS DLSRLTELSLRRRQMLLLETLKVKQTILE PIPTSLKLPIAVSCYWLQHTETKAKLHHL QSLLLTMLVGPLIAIINSPGKEELQEDGA KMLYAEFQRVKAQTRLGTRLDLDTAHI FCQWQSCLQMGDVSQPAAVHSSPRARP NSTVQWKPGARTMPATASIDLCRKSPEH MS 562 A 180 2 TRHFYLKKNNFPPTQKSCLPLFSLSSLCV CVCVCVCVCVCVCVYSSAYKKTRRMHR WIR 563 A 3 301 GLVNMALYVSPIVSGEVIRSRGGSTSEFT PGYVKPKHEVNPQMTLRRLPDEDPQNL ADPAYRRRRIIMQNMRDEELAIAQVEEM QAVSAVLKGKYTMTG 564 A 270 31 SFVVEELSWALQMLGRIPGLFAQIQNLG KEKIFWKEFSILDANKNTDVSWKEIKIAT LTGVWKKLILTFETVSYKSQNQH 565 A 341 3 YDVIVGGELFSDYSDRPRKFVALNPKLK STGAGRYQFLSRWWDAYRKQLGLKDFS PKSQAAVALQQIKERGALPMIDRGDIRQ AIDRCSNMWASLPGAGYGQFEHKADSLI A 566 A 2 265 QEEWSLLSEAQRCLYHDVMLENLTLISS LGCWYGAKDETPSKQTLSIQQESPLRTH WTGGNDVTLKLRLEYTGMVIPHCGLKL LGLK 567 A 376 206 TPGWTQLDSSQVNLYRDEKQENHSSLV SLGKTETLICLKISWHSLRIRTYWAVRSC 568 A 212 428 TQQQAARSSEAHSWQSSSTQSLLEKFFL WRYSRGLRISLWFETESHCVPQVGVRW CDLGSLKPLPPGFRRFS 569 A 3 764 RDKHINNLKKKGQKESEQNREKQQRIET LERYLADLPTLEDHQKQSQQLKDSELKS TELQEKVTELESLLEETQAICREKEIQLES LRQRETEFSSAGHSLQDKQSVEETSGEG PEVEMESWQKRYDSLQKIVEKQQQKMD QLRSQVQSLEQEVAQEEGTSQALREEAQ RRDSALQQLRTAVKELSVQNQDLIEKNL TLQEHLRQAQPGSPPSPDTAQLALELHQ ELASCLQDLQAVCSIVTQRAQGHDPNLS L 570 A 110 498 KVFCQMNSLLDARSLSLTSMLFVCEVCV KMCDPAKGAAGQRTIAALLPCLLDKGM MSTVTEVRALSINTLVKISKSAGAMLKP HAPKLIPALLESLSVLEPQVLNYLSLRAT EQEKAAMDSARLSAAKS 571 A 268 464 ALTSPTLPQPLPGATVIEPLDDISAVTDIL THREGARLETPPPWLAMFTDQPALPNPC SPASVGP 572 A 198 878 GPRQKRKPKSSSSGTKGKGNLEPYLNFL PLSQVNNKFGLRCKNCKTNIHEHCQSYV EMQRCFGFIPPGFHRAYSSPLYSNQQYA CVKDLSAANRNDPVFETLRTGVIMANK ERKKGQADKKNPVAAMMEEEPESARPE EGKPQDGNPEGDKKAEKKTPDDKHKQP GFQQSHYFVALYRFKALEKDDLDFPPGE KITVIDDSNEEWGRGKIGEKVGFFPSNFII WGPA 573 A 120 1 FRPADKTNVKAAWGKVGAHAGEYGAE ALERMFLSFPTTCI 574 A 384 1 RTDSSTTNRESVNSEDGDAKEPPYRGPF CGRDRVHTDFTPSPYDTDSLKLKKGDII DIISKPPMGTWMGLLNNKVGTFYYIYVD VLSEDEEKPKRPTRRRRKGRPPQPKSVE DLLDRINIKEHMPTCI 575 A 460 3 RVVQTVDPSKAQAKPHTLWVAFAKLYE DNGQLDDARVILEKATKVNFKQVDDLA SVWCQCGELELRHENYDEALRLLRKAT ALPARRAEYFDGSEPVQNRVYKSLKVW SMLADLEESLGTFQSTKAVYDRILDLRIA TPHVFGRSEDQASTR 576 A 396 2 CRECTEGEHAELPTVPLKDVVEQHKASL QVQLDAVNKRLPEIDSALQFISEIIHQLT NQKASIVDDIHSTFDELQKTLNVRKSVL LMELEVNYGLKHKVLQSQLDTLLQGQE SIKSCSNFTAQTLNHGTKA 577 A 1 45 LLYTYVLLASGVWVA*AYHRLIENNRN QIIQALLITILLGLYFTLLQASE\HLQSPFTI SDGIYGSTFFVATGFHRLHVIIGSTLLTIC FIPQLIFDFTSKHHFGIQAAV*YWHLVDG V*LFMHGSI*WVA 578 A 2091 320 ISHSLSTQDPNGESIIYNSSFSAVDSFAVS SPASDYLELDTIKNLVKKYSQFINFPIYV WSSKTETVEEPMEEEEAAKEEKEESDDE AAVEEEEEEKKPKTKKVEKTVWDWEL MNDIKPIWQRPSKEVEEDEYKAFYKSFS KESDDPMAYIHFTAEGEVTFKSILFVPTS APRGLFDEYGSKKSDYIKLYVRRVFITD DFHIDMMPKYLNFVKGVVDSDDLPLNVS RETLQQHKLLKVIRKKLVRKTLDMIKKI ADDKYNDTFWKEFGTNIKLGVIEDHSNR TRLAKLLRFQSSHHPTDITSLDQYVERM KEKQDKIYFMAGSSRKEAESSPFVERLL KKGYEVIYLTEPVDEYCIQALPEFDGKR FQNVAKEGVKFDESEKTKESREAVEKEF EPLLNWMKDKALKDKIEKAVVSQRLTE SPWLLWVGQPSTDWSGNMRDFMKAQA YQPGK\DILPN\YYASQK\KTFEIN\PRPPL\ IRDMLRRIK\EDE\DDKTVLDLA\VVLV*N RQRLGSGYLFTQTLKAYGDW/RLERML RLSFEHLTPDAKGGRRAPKEEP*KRQQK DTSRKTQSRDEE/MQEMGWWETDEEEE TSQRNPTA 579 A 25 378 IASGRPFFFFFFFFFFFFFFFFFFFFFFFFFF FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF FFFFFF*FYILKIFFFFFILELCYLSNYGVG VTY/IRMSYYK*MLESYFFFTINIFFIGNN YLISLIYNIYAVKRQL 580 A 1 163 GSTTPAMEFASLFKKILLIDCRD/RGLAL LPRLVLSSWPQVIFLPWPPKFLGLRT 581 A 1 272 ESSCDWLNAYLTLVRCAQD*ADYFAER LYKSMKGAGTDEETLIRIIVTRAEVDLH GIKAKFQEKYQKSLSDMVRSDTFGDFRK LLVALLH 582 A 401 1 LYIFSPSRLIFFRGFSFFFFPPKKIFFLKIFP NDFFFPSHFFKIPSLGFFFYTFFIRKIFFFFP IIYFSPPGFFF*SPPFFFFFFFFFFFFFFFFFF FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFG WYKQRPSQARVWTASLDFTL 583 A 764 139 GDLPDPCKFNFFPIPGPSLSSPPSPWIMFK KFDEKENVSNCIQLKTSVIKGIKNQLIEQ FPGIEPWLNQIMPKKDPVKIVRCHEHIEI LTVNGELLFFRQREGPFYPTLRLLHKYPF ILPHQQVDKGAIKFVLSGANIMCPGLTSP GAKLYPAAVDTIVAIMAEGKQHALCVG VMKMSAEDIEKVNKGIGIENIHYLNDGL WHMKTYK 584 A 1 343 FFFFEDKVSLCHPRWECSDDITAH\CSLN LGFK*SSHL\SLPK*LCLLGTQHYAPANF LYFVFFVETGFRHVGQAGLEHLGSTDPP ASA/FPKCWDYRCEPPCSTRFYYLS*LNF G 585 A 34 475 IPPTMGNLALGGKNSSRGFVDSLICNSSR AFMDWRALLSSLNDFASLSFAESWDNV GLLVEPSPPHTVNTLFLTNDLTDEVMDE VLLKKAHLILSYHPSIFRPMKRITWNTW KELLVIRALENRVGIYSPYTT*EA\APQG VNNWVA 586 A 17 1040 DGVSLLLYRLEYNGVISPHCNPGSSDSPA SASRVAGITGMRHHAQLIFVFLVETGFH HVGQAGLELLTSGDPPASASQSAGITGM SHHAWPDVNYLKNFQVILMGQG\WNPC SGPIRAPGCHKRMHALTPVGLGGFGNVL LLF*DG/CLSVAHPGAQWHDHSSLQPRP PGLR\YPPASAS*VSASGFHDDLGPRHTT LVLFRGQSCTRSPSATAPPRSTDMQS*A ALFPRGSCVGFLGPSVD/YRTPAEDLLG MPHCLCFPSSLNTQSGLAMMPLCLLASP PLEQPSWHATLLAAGHCAFCLYRSAVM LPK*QQQQQQQRLGTWLMPVFPAVRDQ PGQHGETLSL 587 A 236 3 VDHLRSEV*DQPGQHIVKPRLY*KIQKL AGHGGACL*SPLLGKLRQENRLNPG\AR SCSEPRSHYCTPAWGTE*NSVS 588 A 93 413 GYCFSRRSGSMALWRAYQRALAAHPW KVQVLTADFWGGRQHCWPHASSYTCRP RPPGLKD/IASCSSLQEATSCPSAFCA*NS KATKQLVERRGLQEHQRGRTLTMVS 589 A 518 2 VGSQGLVPKKNRPAGKDLGAPSGGPPR KCIP/WQGLLLTAS\LLAL*EAPTTAWLFI ASAPYEVAEGENVHLSVVYLRENLYSY GWYKGKTVEPNQLIAAYVIDTHVRTPGP AYSGRETISPSGDLHFQNVTLEDTGYYN LQVTYRNSQIEQASHHLRVYESVAQPSI QASSCI 590 A 121 473 VTRAHCWKMERSLRQSLLEPVPPSLPNS VLGWC/AELRVLCKLEENVQATNSPSEA EEFQLEVSGLLGEMNCPYLSLTSGDVTK RLLIQKNCLLLLTYLISELEAARMLCVN APPKKS 591 A 243 353 DRVSLLLPRLDCNGTILVHCGLR/LPGFK RFS\CLSLPS 592 A 2 367 QPQTDTMVHLTPEEKAAATVLWGKGN VDEVGGEALVKLIGAYPWTQRFFESFGD LSTPDAVMGNPNGKAHGAKVLGAFSDG LAHLDNLKGTFATLSELHCDKLHVDAE N/FMLLGNVLVCELA 593 A 158 418 IISVKRWMEPGQHGKTPPPLKIQRLAGH/ SQVLRGLRHKNCLNPGDGGCSEPRVFH CTPAWVTEEDPVSLSKKREMDGNNTTF TPSQL 594 A 1 418 PQTQREPTMGLSAAD*TNV*AGWGKVG AHAGEYGAEALERMFLSFPTTKTYFPHF DLSHGYAQDKGHGMKVADALTNAVAH VDDMPNALYALSDLHAHKL*VDPVNFK LLNHCLVVTLACHLLAEVTPGVHAFLD KFPG 595 A 76 440 SYPETWALEDASLEQMDNGYWGYMMT DPVTLNVGGHLYTTILTTLTRYPDSMLG AMNGGDFPTA*DPQGNYFIDLDGPLFRY VLNFLRTSELTLPLDFKEFDLLLK*SDFY QIEPLIQCIN 596 A 83 341 SFRRPMASASTQPAALSAEQAKASIH*FL ALSAGPAVCSPTPTRDASLGCFWRAVVL AEVIQAFSAPENAVRMDEARDNACNDM GK 597 A 102 1 EHRPGVVAHACNPSTSGGQGEWIT*GQE FKTSLG 598 A 180 3 KKKKKKKKRGEIGKFYSLLEELTARSWF SCFFIF*DKQGLPWWLGISYKGSGQYDIQ A 599 A 64 326 GAEVAPLSIYFTQVPVVHIYSVIMTRLVK LAARKQSPFFLAVFGIYHLLITIHF*RQK KKIYLSIYLSIYLSIYLSIYLNDLTEENC 600 A 364 219 NFCTTAS*GRGCSEPR*RHCTPAWVTDY DSISNKN*TGGDFIKNIMRNLQDAIIGSV YFHVERVNRLN 601 A 61 284 EDHHAGTLITALSSH*FFT*VGLEINMLA FIPVLTKKKKKKRGGPFKGTKFKARGGG RKNFFKGAPKLNSGAGV 602 A 3 397 VSEEKLLVRIPRVIGATYSLANERLRALE DIEREIGAILQNAGTVILELSKEKTNERLL DRQAAAFTASVQHVEAELSAQIRYLTQV ATGQPHEGSSYSSRKDCQMALKRVDYA RLKLSDVARTCEQMLEN 603 A 110 370 GTYALKIIGHKRSSEGVQRHHLKGITEIL MNRPSARNALGNVFVSELLETLAQLRED RQVRVLLFRSGVKGVFCAGADLLDREH MS 604 A 110 370 GTYALKIIGHKRSSEGVQRHHLKGITEIL MNRPSARNALGNVFVSELLETLAQLRED RQVRVLLFRSGVKGVFCAGADLLDREH MS 605 A 110 370 GTYALKIIGHKRSSEGVQRHHLKGITEIL MNRPSARNALGNVFVSELLETLAQLRED RQRVLLFRSGVKGVFCAGADLLDREH MS 606 A 42 385 CSSEDKEDSFYPRNSKKKCKDFSKHQVF CFVLRRESCSVARAGVQWRDLGSLQAP PPGFTPFSWKGEDAVSLNRSIAPQPERQS ETLGSNRLRRWRKNYKARRGGHMPVL QVLS 607 A 1 268 RMKTILSNQTVDIPENVDITLKGRTGIVK GPRGTLRRDFNHINVELSLLGKKKKRLR VDKWRGNTKELSTDRTMCIHCHNMIMV VIHVW 608 A 101 3 KHTLGWVQWLMPAIPALWEAKVGRSL ETHASA 609 A 3 299 FHFFRKPPESKKPSVPETEADGFVLLGDT TDEQRMTARGKTSDIEANQPLETNKENS SSVTVSDPEMENKAGQTLENSSLMAELL SDVPVTLAPHVLAG 610 A 283 3 LALFYPQIKAIIFFFYLINASNGSILFFSGP FHTCSLKSSGALWCMIFRGRFHSLVCFS RLSGVLMEVEEHEVLQDSLDRCYSTPPM FFDIPD 611 A 2 363 NSAPEAGSCWKMKVNISFPATGCQKLIE VDDERKLRTFYDNRMAPEVAVDALVEE WKGYVVRIIGGNDKQGFPMKHGDLTHG RVRLLLSKGHSCYRPRRTGERKRKSLRG CIVDANLSVL 612 A 56 415 ALIMSFIFEGIYDGFSSVLQFLGLYKKSG KLVFLGLDNAGKTTLLHMLKDDRLGQH VPTLHPTSEELTIAGMTFTTFDLGGHEQA RRVWKNYLPAINGIVFLVDCADHSRLVE SKVELNA 613 A 32 240 RGIPRLERPNPSLTLAPDTQDLFKLCCKC NKSDPVPPLTRKKKKKKKKKKKKKKKK KKKKKKKKKRGAL 614 A 218 429 ELAPLQSLRKGPGLLSPPRACPVPTPAVS RTLLGNFEESLLRGRFAPSGHIDGYTAEI GASGSYCPQHVT 615 A 67 1204 VPAQPPKSRLSRGELLGLKRPVLVRPRW VGSEQAEATMEQCACVERELDKVLQKF LTYGQHCERSLEELLHYVGQLRAELASA ALQGTPLSATLSLVMSQCCRKIKDTVQK LASDHKDIHSSVSRVGKAIDRNFDSEICG VVSDAVWDAREQQQQILQMAIVEHLYQ QGMLSVAEELCQESTLNVDLDFKQPFLE LNRILEALHEQDLGPALEWAVSHRQRLL ELNSSLEFKLHRLHFIRLLAGGPAKQLEA LSYARHFQPFARLHQREIQVMMGSLVY LRLGLEKSPYCHLLDSSHWAEICETFTR DACSLLGLSVESPLSVSFASGCVALPVL MNIKAVIEQRQCTGVWNHKDELPIEIEL GMKCWYHSVFACPILY 616 A 220 627 VGPKFEPDRSAGRRLAVVFRGNTPSLWL SMSIVTVQLGQCGNQIGFEVFDALLSDS HSSQGLCSMRENEAYQASCKERFFSEEE NGVPIARAVLVDMEPKVINQTLSKAAQS GQWKYGQHACFCQKQGSGNNWAYG 617 A 220 627 VGPKFEPDRSAGRRLAVVFRGNTPSLWL SMSIVTVQLGQCGNQIGFEVFDALLSDS HSSQGLCSMRENEAYQASCKERFFSEEE NGVPIARAVLVDMEPKVINQTLSKAAQS GQWKYGQHACFCQKQGSGNNWAYG 618 A 244 459 CTWNENQRMEIVWEVLFLLQANFIVCIS AQQNSPKIHEGWWAYKEVVQGSFVPVP SFWGLVNSAWNLCSVGK 619 A 258 387 LNYHQNINDFFETESHSVTRLECSGTILA QCNLCLLSPSDSPP 620 A 4 281 RHIVIGEGKLKGQPGASVAAPCVRLVPH SIPVLALSPGLLCSARS\TKQKNSCQQLK EDVD\QTKWGTDTPSMD*ILMEEVKLEE QLKEVVEEYKLALSDTEGLQQSIQKLVD EANMCSIQGFCMDSLEVADVLENGTQC VS*QKNSCQQLKEDVDPNQMGDRYSLY GLDTHGRSQVRRATEGGCGRI 621 A 648 937 RKGQQQTCLS\LRVSWLYRKVTFSIFFFF LIL*K*RWGLTMVTQAGLKLLGTSNPPA FALPKCWDYRCKSLPPAQKLTFSKATTK KTPQICQSMKVS 622 A 3 816 HETMEAAPSRFMFLLFLLTCELAAEVAA EVEKSSDGPGAAQEPTWLTDVPAAMEFI AATEDLEIPAVPILHSMVQKFPGVSFGIS TDSEVLTHYNITGNTICLFRLVDNEQLDL EDEDIESIDATKLSRFIEINSLHMVTEYNP VTVIGLFNSVIQIHLLLINE/PRPPQKYEE NMHRYQKAAKLFQGKIPPLFLVGQL\DE RKMGKVISFFKLKESQLPAL\QFTRL*DD EWDTLPTAEVSVEHVQNFCDGFLSGKLL KENRESEGKTPKVEL 623 A 15 286 DRVSL/LSPRLECSGVVSAHCKLRLPGSR /RFSCLSLS\GSWDYRRPPPRPANFFVFLV ETGFHCGLDLLTS*SARLGLPKCWDYKR EPPHPA 624 A 2411 325 NSSWPAEPAASPWRPLWRALGATFPSGS QPAARTPAGPCIGGMAPPGFKHVSSMLA \LTIIAST\WALTPTHYLTKHDVERLKASL DRPFTNLESAFYS\IVGLSSLG\AQVP\DA KKACTYI\RSNLDP\SNV\DSLFYGWPRAS Q\ALSGM*RSLFSNE\TKDLAFGQLFS*GT SSVYPRSYHAS/VAALKWALGLPLASQE ALSALTARLSKEETVLATVQALQTASHL SQQADLRSIVEEIEDLVARLDELGGVYL Q\FEEGLEFTTAL\FVAATYKA/LMDH\VG TE\PSIKE\DQVIQLMN\AIF\SKKNFE\SLS EAFSV\ASAAAVLSHNRYHVPVVVVPEG SASDTHEQAILRLQVTNVLSQPLTQATV KLEHAKSVAS\RATVLQKTSPTP\VGIVFE LNFMN\VKFS\SGY\YDFLGRKLKGDNRY IS\NTVELRVQDPPTEVGITNVDLSTV\DK DQSIAP\QTTRVTYP\AKAKGTFIADSHQ NFALFFQL\VGVNTGAELTPHQTFVRLH NQKTGQ\EVVFVAEPDNKNVYKFELDTS ERKGLNLTSRSGTYTFY\LIIGDATLKNPI LWNVADVVIKFPEEEAPSTVLSQNLFTP KQEIQHLFREPEKRPPTVVSNTFTALILSP LLLLFALWLRIGANVSNFTFAPSTIIFHLG HAAMLGLMYVYWTQLNMFQTLKYLAI LGSVTFLAGNRMLAQQAVKRTAH 625 A 498 915 QGSKVFIKKCST/SSLTREMQISTQRSHC GTLI*ASEVRK/SG*HQCQR*LWEGDL\L HC**QCELMEPTWKMAWHQLLEKKTHS PL/DLPVPHQGLDMAVM/CYQGA/CARN LYSQILTIAPNWNQPTYPSGREWMLKNA RIAIG 626 A 130 1081 SVSTTRSFSVDSSAKTAAMPVTVTRTTIT TTTTSSSGLGSPMIVGSPRALTQPLGLLR LLQLVSTCVAFSLVASVGAWTGSMGNW SMFTWCFCFSVTLIILIVEL\SGLQARFPL SWRNFPITFACYAALFCLSASIIYPTTYV QFLSHGRSRDHAIAATFFSCIACVAYATE VAWTRARPGEITGYMATVPGLLKVLET FVACIIFAFISDPNLYQHQPALEWCVAVY AICFILAAIAILLNLGECTNVLPIPFPSFLS GLA\FCLSSSMPPPLFSGPSTSSMRSMAA SLGAREM*AAAAAMPTTCVPGTADWL WPS 627 A 1 2059 GCQRFMINMGDSHVDTSSTVSEAVAEE VSLFSMTDMILFSLIVGLLTYWFLFRKK KEEVPEFTKIQTLTSSVRESSFVEKMKKT GRNIIVFYGSQTGTAEEFANRLSKDAHR YGMRGMSADPEEYDLADLSSLPEIDNAL VVFCMATYGEGDPTDNAQDFYDWLQE TDVDLSGVKFAVFGLGNKTYEHFNAMG KYVDKRLEQLGAQRIFELGLGDDDGNL EEDFITWREQFWPAVCEHFGVEATGEES SIRQYELVVHTDIDAAKVYMGEMGRLK SYENQKPPFDAKNPFLAAVTTNRKLNQ GTERHLMHLELDISDSKIRYESGDHV\AV YPANDSALVNQLGKILGADLDVVMSLN NLDEESNKKHPFPCPTSYRTALTYYLDIT NPPRTNVLYELAQYASEPSEQELLRKMA SSSGEGKELYLSWVVEARRHILAILQDC PSLRPPIDHLCELLPRLQARYYSIASSSKV HPNSVHICAVVVEYETKAGRINKGVATN WLRAKEPVGENGGRALVPMFVRKSQFR LPFKATTPVIMVGPGTGVAPFIGFIQERA WLRQQGKEVGETLLYYGCRRSDEDYLY REELAQFHRDGALTQLNVAFSREQSHK VYVQHLLKQDREHLWKLIEGGAHIYVC GDARNMARDVQNTFYDIVAELGAMEH AQAVDYIKKLMTKGRYSLDVWS 628 A 1 1770 MSRRLLPRAEKRRRRLEQRQQPDEQRR RSGAMVKMAAAGGGGGGGRYYGGGSE GGRAPKRLKTDNAGDQHGGGGGGGGG AGAAGGGGGGENYDDPHKTPASPVVHI RGLIDGVVEADLVEALQEFGPISYVVVM PKKRQALVEFEDVLGACNAVNYAADN QIYIAGHPAFVNYSTSQKISRPGDSDDSR SVNSVLLFTILNIPIYSITTDVLYTICNPCG PVQRIVIFRKNGVQAMYEFDSVQSAQRA KASLNGADIYSGCCTLKIEYAKPTRLNV FKNDQDTWDYTNPNLSGQGDPGSNPNK RQRQPPLLGDHPAEYGGPHGGYHSHYH DEGYGPPPPHYEGRRMGPPVGGHRRGP SRYGPQYGHPPPP\PPPPEYGPHADSPVL MVYGLDQSKMNCDRVFNVFCLYGNVE KVKFMKSKPGAAMVEMADGYAVDRAI THLNNNFMFGQKLNYCVSKQPAIMPGQ SYGLEDGSCSYKDFSESRNNRFSTPEQA AKNRIQHPSNVLHFFNAPLEVTEENFFEI CDELGVKRPSSVKVFSGKSERSSSGLLE WESKSDALETLGFLNHYQMKNPNGPYP YTLKLCFSTAQHAS 629 A 1826 5 SNLPEANVMSIVIPLGVDTAETSYLEMA AGSEPESVEASPVVVEKSNSYPHQLYTS SSHHSHSYNGFAPMRTHNRVLVPPPNTP APPPP\SVLISKNEVGHIYPLPNFDETSQC LPTISTS\EDGSYGTDVT\RCICGFTHDDG YMICCDKCSVWQHIDCMGIDRQHIPDTY LCERCQPRNLDKERAVLLQRRKRENMS DGDTSATESGDEVPVELYTAFQHTPTSIT LTASRVSKVNDKRRRKKSGEKEQHISKCK KGSAPEIDPSSDGSNFGWETKIKAWMDR YEEANNNQYSEGVQREAQRIALRLGNG NDKKEMNKSDLNTNNLLFKPPVESHIQK NK\KILKSAK\DWPP\DALIIEYRGKFM\L R\EQFEANGYFF\KRP\YPFVLF\YSKF\HG LEMCVDARTFGNEARFIRRSCTPNAEVR HEIQDGTIHLYIYSIHSIPKGTEITIAFDFD YGNCKYKVDCACLKENPECPVLKRSSES MENINSGYETRRKKGKKDKDISKEKDT QNQNITLDCEGTTNKMKSPETKQRKLSP LRLSVSNNQEPDFIDDIEEKTPISNEVEM ESEEQIAERKRKMNWTVDGCGSWFWFR AEASVKALGLTLNVLFG 630 A 1826 5 SNLPEANVMSIVIPLGVDTAETSYLEMA AGSEPESVEASPVVVEKSNSYPHQLYTS SSHHSHSYNGFAPMRTHNRVLVPPPNTP APPPP\SVLISKNEVGHIYPLPNFDETSQC LPTISTS\EDGSYGTDVT\RCICGFTHDDG YMICCDKCSVWQHIDCMGIDRQHIPDTY LCERCQPRNLDKERAVLLQRRKRENMS DGDTSATESGDEVPVELYTAFQHTPTSIT LTASRVSKVNDKRRKKSGEKEQHISKCK KGSAPEIDPSSDGSNFGWETKIKAWMDR YEEANNNQYSEGVQREAQRIALRLGNG NDKKEMNKSDLNTNNLLFKPPVESHIQK NK\KILKSAK\DWPP\DALIIEYRGKFM\L R\EQFEANGYFF\KRP\YPFVLF\YSKF\HG LEMCVDARTFGNEARFIRRSCTPNAEVR HEIQDGTIHLYIYSIHSIPKGTEITIAFDFD YGNCKYKVDCACLKENPECPVLKRSSES MENINSGYETRRKKGKKDKDISKEKDT QNQNITLDCEGTTNKMKSPETKQRKLSP LRLSVSNNQEPDFIDDIEEKTPISNEVEM ESEEQIAERKRKMNWTVDGCGSWFWFR AEASVKALGLTLNVLFG 631 A 1197 878 LGHCNLSIYSYEIKTGVPSPRGMIYLLLC VCVCVCLPNHAIIVMLNRTEPYVPEGGA YLPEREPFIVPVEPERTAEYEDYGADEPA EEPPEPHRRWR\RALPHGPGQ 632 A 164 418 QIESSSLNVNKKDNHTKTPSEGHQHQRP KVDKSLKMRKKQRKKAENSKNQNASSP PKEHNSSPAKEQN*TENEFDELTEVGFR M 633 A 351 463 MESCSVTQVGVQWHNQLTATPPPRFKR LSS*DYRACI 634 A 88 328 RHKKRCLTYLTIK*MQIKTKMRYHFTHS RMFIVKKDSKCWQICGEIEILTHCWWKC TMVQSLWKIV*PFLKMMEKMRSEF 635 A 434 1142 AQGPYLARHEPLTASSLSRSAMSSEPPPP PQPPTHQASVGLLDTPWSRERSPSPLRG NVVPSPLPTRRTRTFSA*VTGSSGLGSTL NLQGAFLVSHPQPTAQPQVCLFGSPGHR PKNGQRHSESLASGKRSI 636 A 1931 233 FPARPTRPRTRGGVVVGAAAAGPRCGS ARAQVAEGAVRNIQAEGHRQIPDLTWT TLLLLARLPFNSPPLRSGLAPPPDLSLVLL QALTLDLRWLPHLSGAMATGADVRDIL ELGGPEGDAASGTISKKDIINPDKKKSKK SSETLTFKRPEGMHREVYALLYSDKKDA PPLLPSDTGQGYRTVKAKLGSKKVRPW KWMPFTNPARKDGAMFFHWRRAAEEG KDYPFARFNKTVQVPVYSEQEYQLYLH DDAWTKAETDHLFDLSRREDLRFVVIHD RYDHQQFKKRSVEDLKERYYHICAKLA NVRAVPGTDLKIPVFDAGHERRRKEQLE RLYNRTPEQVAEEEYLLQELRKIEARKK EREKRSQDLQKLITAADTTAEQRRTERK APKKKLPQKKEAEKPAVPETAGIKFPDF KSAGVTLRSQRMKLPSSVGQKKIKALEQ MLLELGVELSPTPTEELVHMFNELRSDL VLLYELKQACANCEYELQMLRHRHEAL ARAGVLGGPATPASGPGPASAEPAVTEP GLGPDPKDTIIDVVGAPLTPNSRKRRESA SSSSSVKKAKKP 637 A 1931 233 FPARPTRPRTRGGVVVGAAAAGPRCGS ARAQVAEGAVRNIQAEGHRQIPDLTWT TLLLLARPFNSPPLRSGLAPPPDLSLVLL QALTLDLRWLPHLSGAMATGADVRDIL ELGGPEGDAASGTISKKDIINPDKKKSKK SSETLTFKRPEGMHREVYALLYSDKKDA PPLLPSDTGQGYRTVKAKLGSKKVRPW KWMPFTNPARKDGAMFFHWRRAAEEG KDYPFARFNKTVQVPVYSEQEYQLYLH DDAWTKAETDHLFDLSRRPDLRFVVIHD RYDHQQFKKRSVEDLKERYYHICAKLA NVRAVPGTDLKIPVFDAGHERRRKEQLE RLYNRTPEQVAEEEYLLQELRKIEARKK EREKRSQDLQKLITAADTTAEQRRTERK APKKKLPQKKEAEKPAVPETAGIKFPDF KSAGVTLRSQRMKLPSSVGQKKIKALEQ MLLELGVELSPTPTEELVHMFNELRSDL VLLYELKQACANCEYELQMLRHRHEAL ARAGVLGGPATPASGPGPASAEPAVTEP GLGPDPKDTIIDVVGAPLTPNSRKRRESA SSSSSVKKAKKP 638 A 390 1258 LTRAPPPSTQVRPVPGPPAGAVVAAPGG ALASVSFDSRDSKMAAQSAPKVVLKSTT KMSLNERFTNMLKNKQPTPVNIRASMQ QQQQLASARNRRLAQQMENRPSVQAAL KLKQSLKQRLGKSNIQARLGRPIGALAR GAIGGRQLPIIQRGLPRGGLRGGRATRTL LRGGMSLRGQNLLRGGRAVAPRMGLRR GGVRGRGGPGRGGLGRGAMGRGGIGG RGRGMIGRGRGGFGGRGRGRGRGRGAL ARPVLTKEQLDNQLDAYMSKTKGHLDA ELDAYMAQTDPETND 639 A 1727 834 RPAEPDPGSGHPEPKSPTLSAVMSAEVK VTGQNQEQFLLLAKSAKGAALATLIHQ VLEAPGVYVFGELLDMPNVRELAESDF ASTFRLLTVFAYGTYADYLAEARNLPPL TEAQKNKLRHLSVVTLAAKVKCIPYAV LLEALALRNVRQLEDLVIEAVYADVLRG SLDQRNQRLEVDYSIGRDIQRQDLSAIAR TLQEWCVGCEVVLSGIEEQVSRANQHK EQQLGLKQQIESEVANLKKTIKVTTAAA AAATSQDPEQHLTELREPAPGTNQRQPS KKASKGKGLRGSAKIWSKSN 640 A 56 443 RLHVVPMARYEEVSVSGFEEFHRAVEQ HNGKTIFAYFTGSKDAGGKSWCPDCVQ AEPVVREGLKHISEGCVFIYCQVGEKPY WKDPNNDFRKNLKVTAVPTLLKYGTPQ KLVESECLQANLVEMLFSED 641 A 320 43 LFYEEFSYATILTISSSEQKNSIKKSTFCLT LLPRLECSGAVMARCSLNLLGSGDPPAS ASQVAGTTGACHHTSLIFPFVLYVDSTG LTLKT 642 A 45 400 DPRVRTREWDSLNYENHIYLMPFIYINII LGGTISLLGRLVYRSHLISSLLCLEGIILSL FIIATLITLNTHSLLTNIGPIAILVFAACEA AGGLSLLGSISNTYGLNYVHNLNLLQC 643 A 3828 2909 AECEEVRRKSELFNPVSLDCKLRQKAIA EVDVGTDKAQNSDPILDTSSLVPGCSSV DNIKSSQTSQNQGLGRPTLEGDEETSEVE YTVNKGPASSNRDRVPPSSEASEHHPRH RVSSQAEDTSSSFDNLFIDRLQRITIADQ GEQQSEENASTKNLTGLSSGTEKKPHYM EVLEMRAKNPVPQLRKFKTNVLPFRQN DSSSHCQKSGSPISSEERRRRDKQNLDDI TAARLLPLHHMPTQLLSIEESLALQKQQ KQNYEEMQAKLAAQKLAERLNIKMRSY NPEGESSGRYREVRDEDDDWSSDEF 644 A 3 2185 RPYLGLLKMAALEEEFTLSSVVLSAGPE GLLGVEQSDKTDQFLVTDSGRTVILYKV SDQKLPLGSWSVKQGQIITCPAVCNFQTG EYVVVHDNKVLRIWNNEDVNLDKVFK ATLSAEVYRILSVQGTEPLVLFKEGAVR GLEALLADPQQKIETVISDEEVIKWTKFF VVFRHPVLIFITEKHGNYFAYVQMFNSRI LTKYTLLLGQDENSVIKSFTASVDRKFIS LMSLSSDGCIYETLIPIRPADPEKNQSLV KSLLLKAVVSGNARNGVALTALDQDHV AVLGSPLAASKECLSVWNIKFQTLQTSK ELPQGTSGQLWYYGEHLFMLHGKSLTVI PYKCEVSSLAGALGKLKHSQDPGTHVV SHFVNWETPQGCGLGFQNSEQSRRILRR RKIEVSLQPEVPPSKQLLSTIMKDSEKHIE VEVRKFLALKQTPDFHTVIGDTVTGLLE RCKAEPSFYPRNCLMQLIQTHVLSYSLC PDLMEIALKKKDVQLLQLCLQQFPDIPES VTCACLKIFLSIGDDSLQETDVNMESVF DYSINSVHDEKMEEQTEILQNGFNPEED KCNNCDQELNKKPQDETKESTSCPVVQ KRAALLNAILHSAYSETFLLPHLKDIPAQ HITLFLKYLYFLYLKCSENATMTLPGIHP PTLNQIMDWICLLLDANFTVVVMMPEA KRLLINLYKLVKSQISVYSELNKIEVSFR ELQKLNQEKNNRGLYSIEVLELF 645 A 118 473 WWPVLRRWTMKTFRWKVKPGMDVAS VPSVRKVRFGDGYSQRAPAGLNANLKT YSVTLSVPREEATVLESFLEEHGGWKSF LWTPPYEWRQIKVTCAKWSSRVSMLRV EFSAEFEQVVN 646 A 138 632 QSVWVAQPPAVTADLQFLEGSGEKNAP TAVLRKRQEEMRPLDIDEVEAPEEVEVL EPEEDFEQFLLPVINEMREDIASLIREHGR AYLRTRSKLWEMDNMLIQIKTQVEASEE SALNHVQHPSGEADERVSELCEKAEEKA KEIAKMAEMLVELVWRIERSESS 647 A 2224 1709 QASGLYSFLTLAPMTHLLLTATVTPSEQ NSSREPGWETAMAKDILGEAGLHFDEL NKLRVLDPEVTQQTIELKEECKDFVDKI GQFQKIVGGLIELVDQLAKEAENEKMK AIGARNLLKSIAKQREAQQQQLQALIAE KKMQLERYRVEYEALCKVEARQNEFID QFIFQK 648 A 84 816 TGNKMQDPNADTEWNDILRKKGILPPK ESLKELEEEAEEEQRILQQSVVKTYEDM TLEELEDHEDEFNEEDERAIEMYRRRRL AEWKATKLKNKFGEVLEISGKDYVQEV TKAGEGLWVILHLYKQGIPLCALINQHL SGLARKFPDVKFIKAISTTCIPNYPDRNLP TIFVYLEGDIKAQFIGPLVFGGMNLTRDE LEWKLSESGAIMTDLEENPKKPIEDVLLS SVRRSVLMKRDSDSEGD 649 A 62 413 MTRQEELAAARAALHDLMTGKRVATV QKDGRRVEFTATSVSDLKKYIAELEVQT GMTQRRTGPAGFYV 650 A 196 339 RSYFPYSPGMFGDDCHQLCDCEGETFCH PKTEKCLCPRGRTGARCDAG 651 A 1280 906 LRMTTATRQEVLGLYRSIFRLARKWQA TSGQMEDTIKEKQYILNEARTLFRKNKN LTDTDLIKQCIDECTARIEIGLHYKIPYPR PIHLPPMGLTPLRGRGLRSQEKLRKLSKP VYLRSHDEVS 652 A 1359 39 CDAPLGGSIAGPFDQLEEILGWESAAAH SAELVAKGTVGKRKWGCAGASSSGSAL LPPCRELLMGHRFLRGLLTLLLPPPPLYT RHRMLGPESVPPPKRSRSKLMAPPRIGT HNGTFHCDEALACALLRLLPEYRDAEIV RTRDPEKLASCDIVVDVGGEYDPRRHRY DHHQRSFTETMSSLSPGKPWQTKLSSAG LIYLHFGHKLLAQLLGTSEEDSMVGTLY DKMYENFVEEVDAVDNGISQWAEGEPR YALTTTLSARVARLNPTWNHIPDQDTEA GFKRAMDLVQEEFLQRLDFYQHSWLPA RALVEEALAQRFQVDPSGEIVELAKGAC PWKEHLYHLESGLSPPVAIFFVIYTDQAG QWRIQCVPKEPHSFQSRLPLPEPWRGLR DEALDQVSGIPGCIFVHASGFIGGHRTRE GALSMARATLAQRSYLPQIS 653 A 3 1652 QNAKVGSEAIQVLPAFPREVQRLPEPAK SCRQRQLEARKAAAEKKKIQKEKLSTPE KIKQEALELAGITSDPGLSLKGGLSQQGL KPSLKVEPQNHFSSFKYSGNAVVESYSV LGNCRPSDPYSMNSVYSYHSYYAQPSLT SVNGFHSKYALPSFSYYGFPSSNPVFPSQ FLGPGAWGHSGSSGSFEKKPDLHALHNS LSPAYGGAEFAELPSQAVPTDAHHPTPH HQQPAYPGPKEYLLPKAPLLHSVSRDPS PFAQSSNCYNRSIKQEPVDPLTQAEPVPR DAGKMGKTPLSEVSQNGGPSHLWGQYS GGPSMSPKRTNGVGGSWGVFSSGESPAI VPDKLSSFGASCLAPSHFTDGQWGLFPG EGQQAASHSGGRLRGKPWSPCKFGNST SALAGPSLTEKPWALGAGDFNSALKGSP GFQDKLWNPMKGEEGRIPAAGASQLVF YQHKNLNQPNHGLALWEAKMKQLAER ARARQEEAARLGLGQQEAKLYGKKRK WGGTVVAEPQQKEKKGVVPTRQALAV PTDSAVTVSSYAYTKVTGPYSRWI 654 A 653 2 KYRWGFTVLARMVSIS*SRDLPVSASQS AGIIGVSHRARPRFYFLISLWHLLYFLIFF QS*Q*MKNLCLKKLPDDSNYCSCTTLFK/ TGNLSCLPDILCTKS*YSVS/CKSFANSYI QKKHDKM*KEGVR*GIINNLRLSIILILI LIS*LSYFNDFH*NSGTSLIVS*HYYLNKH DLHPNYFILLFCFSF/VRQGLRSVAQAGV QWCDLGSLEPPPPRF 655 N 415 0 SKSPPPKRKLFPSSPPKTFYPPPHSGVFPP FPP*NFFFSPRGLIFGGGFFQFFPPPKKRFF SKIPEAFLKVPPKRKKIVFATAPV/YFGPP REFFKGPPP 656 A 429 0 PSSSSPFFFFFPPPQKGIFFPPFFFCFPRVFS PPPFFIPPPRIFFLGPLKKKKTPPPPGGKNF FF*RAPPSP 657 A 222 140 GYWGCAIGKARQAAKTEIEKLQVIINA 658 A 30 250 YPMFQVQPVQHVYPAQVQYVEGGDAV YTNGAMCVLLPRATPGTLSLTPAFATW GGIKQIPLPYAFGDVFTKYC 659 A 1 707 HRPDGCPDIGPTGELSGSLKIPNRDSGIDS PSSSVAGENFPCEEGLEAGPSPTVLGAH AEMALDSQVPKVTPQEEADSDVGEEPD SENTPQKADKDAGLAQHSGPQKLLHIA QELLHTEETYVKRLHLLDQVFCTRLTDA GIPPEVIMGIFSNISSIHRFHGQFLLPELKT RITEEWDTNPRLGDILQKLAPFLKMYGE YVKNFDRAVGLVSTWTQRSPLFKDVVH SIQVRRRRG 660 A 379 3 MAEMQLAELRAEIKHFVSERKYDEELG KAARFSCDIEQLKAQIMLCGEIPHPKNN YSSRTPCSSLLPLLNAHAATSGKQSNFSR KSSTHNKPSEGKAANPKMVSSLPSTADP SHQTMTANKQNGE 661 A 379 3 MAEMQLAELRAEIKHFVSERKYDEELG KAARFSCDIEQLKAQIMLCGEIPHPKNN YSSRTPCSSLLPLLNAHAATSGKQSNFSR KSSTHNKPSEGKAANPKMVSSLPSTADP SHQTMTANKQNGE 662 A 3 783 TLQTLAPATVQTVAAPQVQQVPVSGWK GFREALVGAVNWSLVEDVVFMAAVQN PALTALTTPIQTAALQVPVRAAFSPTLLP PPLFLEVLVFRDVKQVPGGVKQLEPPKE GERRTT\HNIIEKRIRSS\INDKIIELKDLV MGTDAKVGAKQNCVLCSTISPLPVFASG A*RSPRLKVSEYKTELEVLFY*SILVSCQ DYKILKNIRDW*GLIRQ*PAETFDSFGYA LQLMGTPLSEAEKDACHSVVPDTGLCVP WSQLMA 663 A 346 28 YITPGICPSLSTMKALVECAGGKVLSKQP SFRKLMEHKQNSVGRVESASESVIALYH IQRSVTEKLIFFSL\SLSEIILISCENDLHLC REYFARGIGTFPRLPV 664 A 558 175 ASLSCGGLHPVRASWLLCLTKQAWAME GAPPPASLPPCSLISDCCASNQRDSVGVG PSEPCAGYNLVVRRFLSRSEKRNIRVGV TRFSRCV/LSPLSLTQKGNSLTPCASQAR QCLALLRLAHGARTH 665 A 3 377 GRVRLL/NNWDVCANMFGTFFDTEDPG EFKMKYWGVTSFLQKGNDDHWIVDTD YDTYAGQYSCRLLNLDGTCADSYSFVFS RDPNGLPPEAQKIVRQRQEELCLARQYR LIVHNGYCDGRSKRNLL 666 A 355 250 QAGLELLTSDDLPASASKSAGTTGVSHR ARPTLVF 667 A 2 118 GNVLPVLYGEMRVGSRVVSQEISTADEG DGGQVVVIGR 668 A 3 339 TEQKSKSKQHRVSRRAQQRAESPESSAI ESTQSTPQKGRGRPSKTPSPSQPKKNVR VGRSKQAATKENDSSEEVNVFQGSSPVD DIPQEETEEEEVSTVNVRRRSAKRERR 669 A 2 120 NYRRRPRPPNAPSQDGKEAKAGEAPTEN PAPPTQQSSAE 670 A 2 120 NYRRRPRPPNAPSQDGKEAKAGEAPTEN PAPPTQQSSAE 671 A 3 284 KTENDHINLKVAGQDGSLVQFKIKRRTP LSKLMKAYCERQGLSMRHITLRFDGQSI NETDTPAQLEMEDEDTIDVFQQQTESVP DSILAGHSF 672 A 2 439 ERVLENVEHYQELKKMVQQSDLGQYVT FLRSFSDKQKISLLHSCTCVLYTPSNEHF GIVPLEAMYMQCPVIAVNSGGPLESIDH SVTGFLCEPDPVHFSEAIEKFIREPSLKAT MGLAGRARVKEKFSPEAFTEQLYRYVT KLAG 673 A 1 631 KADFMRPRISFVDRCYIFSMLDNEVGAN PRGGHQCGSALSCDEGVPAAQGASSEKP HECTKCGKALCCRSDLRVHHGVHAGEK SSACSERGSGFREKLCPDKQGTHTKEKP ARDSRSGKTIFRKTRLCVPGTVHAGAKP YKCWECEKTSHKSRLIEHLRSHTGEKPC GCRECGKAFFQKSHLILRQRTHTGEKPC GCAECGKAAPRTPAS 674 A 3 242 VWPLPCRDADAQSEGINASARYPKNWV TTGDPAREFTMIHSAPLMLLADPDEFES VQLAQSWPLGAIVSLWRSPCRKMN 675 A 3 501 RGININKKDVHTETPCKGHQHQRPKVD KSMKMRKS*CKKAENSKNQNGSSPAKD YNSSPAREQNWMENWLDKLTEVGFIRW IITNSFKLKEHVLTQCKELRS/FEKKLEEL LTKIISLEKTISV*GEG*IQILDRSL*RVCV GYLRRSC 676 A 1 405 AAAAAADMERQEESLSARPALETEGLRF LHTTEGSLLATYGWNIVFSCILLYVVFQ KLSARLRALRHRQLDRAAAAVEPDVDV KLQEALAAARLKMQEELNAQVEKHKE KLKQLE*EKRRQTIEMWDSMQDGKSY 677 A 588 3 GGWLAFPLACPGRCHQPPAAYASAALL NLGRPEAPAHPQTAGSVRPAAVPPGC*G RTGGAGSPAGHSATGPAAPPPRGSTSSA PPARQAEAGLESSSAQVIRPPRSSKVLGL QA*ATAPGLPQLSEGDPGFPSGRVTWIG QSPASEDSWRPSAVSSLTYSPVSPARGC RSPS*KSGKT*KSRQPKRENPPGTAP 678 A 98 314 ILKAQATVGISINLDSPFTSPPLREEIMAN NFSLESHNISLTEHSSMPVENNITLERPSN VNLTCQFTTSGD 679 A 386 545 GNMKKQGISNFISYIYTHTHTHTHTHTYI YIYIHIYTYTHTYNFFFLERELHS 680 A 311 89 RRPECVWGRRGPAGEESEQTALIPLPRG LLTPPNWSGPPGLPPAASQPIILPGPGPSP NLLKSSTSSERSRGAK 681 A 860 84 PWVLTMNFSGGGRQEAAGSRGRRAPRP REQDRDVQLSKALSYALRHGALKLGLP MGADGFVPLGTLLQLPQFRGFSAEDVQ RVVDTNRKQRFALQLGDPSTGLLIRANQ GHSLQVPKLELMPLETPQALPPMLVHGT FWKHWPSILLKGLSCQGRTHIHLAPGLP GDPGIISGMRSRCEIAVFIDGPLALADGIP FFRSANGVILTPGNTDGFLLPKYFKEALQ LRPTRKPLSLAGDEETECQSSPKHSSRER RRIQQ 682 A 420 22 FDQPPGLKATRDEKDSLLNIARGKKNGE KTRRVSSRKKPALKATSDEKDSFSNITR GKKDGEISRKVSSQKPPTLKGTSDEEDS VLGIARENKDGEKSRTVSSEKPPGLKAS SDEKENVLNIARVYCVRCRGS 683 A 1248 103 EALHLAHFGLTGFTQTHCARAWGGQTR KRRRVPRDPGGANCAGGGVSADTSGLA AAARGPPTLGFRERRSARSLDRIRPILVL MVDKKLVVVFGGTGAQGGSVARTLLE DGTFKVRVVTRNPRKKAAKELRLQGAE VVQGDQDDQVIMELALNGAYATFIVTN YWESCSQEQEVKQGKLLADLARRLGLH YVVYSGLENIKKLTAGRLAAAHFDGKG EVEEYFRDIGVPMTSVRLPCYFENLLSHF LPQKAPDGKSYLLSLPTGDVPMDGMSV SDLGPVVLSLLKMPEKYVGQNIGLSTCR HTAEEYAALLTKH\PARSCTMYR*LLRTT KSLAFPVPGTW/LNMFRFYALRPDRDIEL TLRLNPKALTLDQWLEQHKGDFNLL 684 A 148 2357 PNSMVVEHPEFLKAGKEPGLQIWRVEKF DLVPVPTNLYGDFFTGDAYVILKTVQLR NGNLQYDLHYWLGNECSQDESGAAAIF TVQLDDYLNGRAVQHREVQGFESATFL GYFKSGLKYKKGGVASGFKHVVPNEVV VQRLFQVKGRRVVRATEVPVSWESFNN GDCFILDLGNNIHQWCGSNSNRYERLKA TQVSKGIRDNERSGRARVHVSEEGTEPE AMLQVLGPKPALPAGTEDTAKEDAANR KLAKLYKVSNGAGTMSVSLVADENPFA QGALKSEDCFILDHGKDGKIFVWKGKQ ANTEERKAALKTASDFITKMDYPKQTQ VSVLPEGGETPLFKQFFKNWRDPDQTDG LGLSYLSSHIANVERVPFDAATLHTSTA MAAQHGMDDDGTGQKQIWRIEGSNKV PVDPATYGQFYGGDSYIILYNYRHGGRQ GQIIYNWQGAQSTQDEVAASAILTAQLD EELGGTPVQSRVVQQKEPAHLMSLFGG KPMIIYKGGTSRRGGQTAPASTRLFQVR ANSAGATRAVEVLPKAGALNSNDAFVL KTPSAAYLWVGTGASEAEKTGAQELLR VLRAQPVQVAEGSEPDGFWEALGGKAA YRTSPRLKDKKMDAHPPRLFACSNKIGR FVIEEVPGELMQEDLATDDVMLLDTWD QVFVWVGKDSQEEEKTEALTSAKRYIET DPANRDRRTPITVVKQGFEPPSFVGWFL GWDDDY\WSVGPLGTGPMAGAGLP 685 A 361 570 NCFLVFSPARSTVGEFASMSSEECI*MHV VFLFPLTLCVVSL*ERQEAEKMFKGKRG AQLAKDIARRSKT 686 A 2484 1122 QAQPMGRVGGMAQPMGRAGAPKPMG RAGSARRGRFKGCWSEGSPVHPVPAVL SWLLALLRCASTMLSLRVPLAPITDPQQ LQLSPLKGLSLVDKENTPPALSGTRVLA SKTARRIFQEPTEPKTKAAAPGVEDEPLL RENPRRFVIFPIEYHDIWQMYKKAEASF WTAEEVDLSKDIQHWESLKPEERYFISH VLAFFAASDGIVNENLVERFSQEVQITEA RCFYGFQIAMENIHSEMYSLLIDTYIKDP KEREFLFNAIETMPCVKKKADWALRWI GDKEATYGERVVAFAAVEGIFFSGSFASI FWLKKRGLMPGLTFSNELISRDEGLHCD FACLMFKHLVFLKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVA DRLMLELGFSKVFRVENPFDFMENISLE GKTNFFEKRVGEYQRMGVMSSPTENSF TLDADF 687 A 25 219 RNMAAATLTSKLYSLLFRRTSTFALTIIV GVMFFERAFDQGADAIYDHVNEGKLW KHIKHKYENK 688 A 1 1034 QWRNSSHRCLLLHPPLRRLPPALPPPPPP CCCSKLLPPLGLKGFQMEHFDASLSTYF KALLGPRDTRVKGWFLLDNYIPTFICSVI YLLIVWLGPKYMRNKQPFSCRGILVVYN LGLTLLSLYMFCELVTGVWEGKYNFFC QGTRTAGESDMKIIRVLWWYYFSKLIEF MDTFFFILRKNNHQITVLHVYHHASMLN IWWFVMNWVPCGHSYFGATLNSFIHVL MYSYYGLSSVPSMRPYLWWKKYITQGQ LLQFVLTIIQTSCGVIWPCTFPLGWLYFQI GYMISLIALFTNFYIQTYNKKGASRRKD HLKDHQNGSMAAVNGHTNSFSPLENNV KPRKLRKD

TABLE 7 SEQ ID Position of end of signal MaxS MeanS NO: in amino acid sequence (maximum score) (mean score) 1 29 0.962 0.801 5 17 0.989 0.926 15 33 0.933 0.780 20 39 0.899 0.608 23 18 0.992 0.867 25 32 0.928 0.768 26 37 0.979 0.808 27 48 0.977 0.661 28 18 0.965 0.805 29 16 0.972 0.860 30 21 0.948 0.785 31 23 0.954 0.824 32 18 0.966 0.896 33 15 0.989 0.956 39 16 0.988 0.971 61 37 0.979 0.808 62 21 0.990 0.901 73 48 0.977 0.661 86 18 0.965 0.805 87 16 0.949 0.749 88 16 0.949 0.749 89 16 0.949 0.749 91 16 0.972 0.860 102 21 0.948 0.785 103 19 0.893 0.604 104 28 0.945 0.556 106 17 0.965 0.869 107 16 0.980 0.957 108 22 0.969 0.917 115 23 0.954 0.824 126 18 0.966 0.896 138 38 0.901 0.607 139 33 0.976 0.754 140 27 0.966 0.909 147 38 0.901 0.607 149 22 0.929 0.838 162 32 0.949 0.704 168 27 0.966 0.909 171 42 0.895 0.609 

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-172, or 345-516, a mature protein coding portion of SEQ ID NO: 1-172, or 345-516, an active domain coding portion of SEQ ID NO: 1-172, or 345-516, and complementary sequences thereof.
 2. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.
 3. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim
 1. 4. The polynucleotide of claim 1 wherein said polynucleotide is DNA.
 5. An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.
 6. A vector comprising the polynucleotide of claim
 1. 7. An expression vector comprising the polynucleotide of claim
 1. 8. A host cell genetically engineered to comprise the polynucleotide of claim
 1. 9. A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.
 10. An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-172, or 345-516.
 11. A composition comprising the polypeptide of claim 10 and a carrier.
 12. An antibody directed against the polypeptide of claim
 10. 13. A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.
 14. A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.
 15. The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.
 16. A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.
 17. A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
 18. A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
 19. A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from SEQ ID NO: 1-172, or 345-516, a mature protein coding portion of SEQ ID NO: 1-172, or 345-516, an active domain coding portion of SEQ ID NO: 1-172, or 345-516, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-172, or 345-516, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).
 20. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of any one of the polypeptides SEQ ID NO: 173-344, or 517-688, the mature protein portion thereof, or the active domain thereof.
 21. The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.
 22. A collection of polynucleotides, wherein the collection comprising the sequence information of at least one of SEQ ID NO: 1-172, or 345-516.
 23. The collection of claim 22, wherein the collection is provided on a nucleic acid array.
 24. The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.
 25. The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.
 26. The collection of claim 22, wherein the collection is provided in a computer-readable format.
 27. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.
 28. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier. 